Compositions for inactivating pathogenic microorganisms, methods of making the compositions, and methods of use thereof

ABSTRACT

Nanoemulsion compositions with low toxicity that demonstrate broad spectrum inactivation of microorganisms or prevention of diseases are described. The nanoemulsions contain an aqueous phase, an oil phase comprising an oil and an organic solvent, and one or more surfactants. Methods of making nanoemulsions and inactivating pathogenic microorganisms are also provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 11/067,626, which is a continuation of U.S. patent applicationSer. No. 10/860,582, which claims the benefit under 35 USC § 119(e) ofU.S. application Ser. No. 60/475,633, filed Jun. 4, 2003, incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods fordecreasing the infectivity, morbidity, and/or rate of mortalityassociated with a variety of pathogenic microorganisms.

BACKGROUND OF THE INVENTION

Pathogenic microorganisms such as bacteria, fungi, viruses, andbacterial spores are responsible for a plethora of human and animalailments. In addition to vegetatively growing bacteria, bacteria of theBacillus genus and others form stable spores that resist harshconditions and extreme temperatures. For example, contamination offarmlands with B. anthracis can lead to a fatal disease in domestic,agricultural, and wild animals, as well as in humans in contact withinfected animals or animal products. B. anthracis infection in humans isno longer common due to effective animal controls that include vaccines,antibiotics, and appropriate disposal of infected livestock. However,animal anthrax infection still represents a significant problem due tothe difficulty of decontaminating land and farms. Moreover, B. anthracisspores can be used as a biological weapon. While an anthrax vaccine isavailable and can be used for the prevention of classic anthrax, geneticmixing of different bacterial strains can render it ineffective.

Other members of the Bacillus genus are also reported to be etiologicalagents for many human diseases. B. cereus is a common pathogen involvedin food borne diseases due to the ability of the spores to survivecooking procedures. It is also associated with local sepsis, wound andsystemic infection.

Although antibiotic and antimicrobial therapy is very effective and amainstay of modern medicine, these therapies suffer from severaldisadvantages. For example, bacterial strains can develop antibioticresistance. A person infected with an antibiotic resistant strain ofbacteria faces serious and potentially life-threatening consequencesbecause antibiotics cannot eliminate the infection. Pneumococci, whichcause pneumonia and meningitis, Salmonella and E. coli which causediarrhea, and enterococci which cause blood stream, surgical wound, andurinary tract infections can all develop antibiotic resistance resultingin fatal infections.

Moreover, antibiotics are not effective in eliminating or inactivatingbacterial spores and viruses. Disinfectants and biocides, such as sodiumhypochlorite, formaldehyde and phenols can be effective againstbacterial spores, but are not well suited for decontamination of theenvironment, equipment, or casualties. The toxicity of these compoundscan result in tissue necrosis and severe pulmonary injury followingcontact or inhalation of volatile fumes. Furthermore, the corrosivenature of commonly used disinfectants and biocides renders themunsuitable for decontamination of sensitive equipment.

Viruses are additional pathogens that infect human and animals whichcurrently lack effective means of inactivation. For example, influenza Avirus is a common respiratory pathogen widely used as a model system totest anti-viral agents in vitro and in vivo. The envelope glycoproteinsof influenza A, hemagglutinin (HA) and neuraminidase (NA), whichdetermine the antigenic specificity of viral subtypes, mutate readily,rendering antibodies incapable of neutralizing the virus. Currentanti-viral compounds and neuraminidase inhibitors are minimallyeffective and viral resistance is common.

SUMMARY OF THE INVENTION

Accordingly, there remains a need in the art for anti-pathogeniccompositions and methods that decrease the infectivity, morbidity,and/or mortality associated with pathogenic exposure while minimizingmicrobial resistance, toxicity to the recipient, and deleterious effectsto equipment and the environment.

To address these and other needs, the present invention providesemulsions comprising an aqueous phase, an oil phase comprising an oiland an organic solvent, and at least one surfactant. The emulsioncomprises particles preferably having an average diameter of less than150 nm.

In one embodiment, the invention provides a method of reducing theaverage nanoemulsion particle size of a composition comprising ananoemulsion, comprising treating a nanoemulsion comprising an aqueousphase, an oil phase comprising an oil and an organic solvent, and asurfactant, and having nanoemulsion particles of an average diameter ofgreater than or equal to about 250 nm, so as to reduce the averagediameter of the nanoemulsion particles to less than 150 nm.

In another embodiment, the invention provides a method of making ananoemulsion, comprising passing a first nanoemulsion through a highpressure homogenizer or a microfluidizer under conditions effective toreduce the average diameter of the nanoemulsion particles less than 150nm. The nanoemulsion comprises an aqueous phase, an oil phase comprisingan oil and an organic solvent, and one or more surfactants. The initialnanoemulsion particles have an average diameter of greater than or equalto about 250 nm.

The invention also provides a method of inactivating a microorganism,comprising contacting the microorganism with a composition comprising ananoemulsion for a time effective to inactivate the microorganism. Thenanoemulsion comprises an aqueous phase; an oil phase comprising an oiland an organic solvent and one or more surfactants. The nanoemulsionparticles have an average diameter of less than 150 nm.

The invention further provides a method of inactivating a pathogenicmicroorganism comprising contacting a subject infected with themicroorganism with a composition comprising a nanoemulsion. Thenanoemulsion comprises an aqueous phase, an oil phase comprising an oiland an organic solvent, and one or more surfactants, wherein thenanoemulsion comprises particles having an average diameter of less than150 nm.

In an additional embodiment, the invention provides an immunogeniccomposition comprising a nanoemulsion, wherein the nanoemulsioncomprises an aqueous phase, an oil phase comprising an oil and anorganic solvent, and a surfactant, wherein the nanoemulsion comprisesnanoemulsion particles having an average diameter of less than or equalto about 250 nm and a microorganism or a portion thereof.

In a further embodiment, the invention provides method of vaccinatingagainst a microorganism comprising administering to a subject the acomposition comprising a nanoemulsion, wherein the nanoemulsioncomprises an aqueous phase, an oil phase comprising an oil and anorganic solvent, and one or more surfactants, wherein the nanoemulsioncomprises particles having an average diameter of less than or equal toabout 250 nm. The microorganism is inactivated by the composition and animmunological response by the subject is elicited.

In another embodiment, the invention provides a method of preventing aninfected state caused by a microorganism, comprising administering to asubject, either before or after exposure to a microorganism, acomposition comprising a nanoemulsion. The nanoemulsion comprises anaqueous phase, an oil phase comprising an oil and an organic solvent,and one or more surfactants, wherein the nanoemulsion comprisesparticles having an average diameter of less than 150 nm.

The invention further provides a kit comprising a composition comprisinga nanoemulsion, wherein the composition is provided in a singleformulation or a binary formulation, wherein the binary formulation ismixed prior to using the composition.

The above described and other features are exemplified by the followingfigures and detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Average separation of neat (100%) emulsions stored at 55° C.

FIG. 2. Average settling of 10% emulsions stored at 55° C.

FIG. 3. Average settling of 2.5% emulsions stored at 55° C.

FIG. 4. Change in pH after accelerated stability testing. pH of neat anddiluted emulsions is measured on day 0 and after 31 days incubation at55° C.

FIG. 5. Dependence of nanoemulsion particle size of passage number andpressure in Avestin EmulsiFlex® C3.

DETAILED DESCRIPTION

Unless otherwise specified, “a” or “an” means “one or more”. The presentinventors discovered that compositions having emulsion particles with anaverage particle diameter of less than 250 nm (“small particle sizenanoemulsion”) have improved stability and/or activity. These smallparticle size nanoemulsions are useful in a wide range of applicationsfor decreasing the infectivity, morbidity, and/or rate of mortalityassociated with a variety of pathogenic microorganisms. As used herein,the term “pathogenic microorganism” refers to a biological microorganismthat is capable of producing an undesirable effect upon a host animal,and includes, for example, without limitation, bacteria, viruses,bacterial spores, molds, mildews, fungi, and the like. This includes allsuch biological microorganisms, regardless of their origin or of theirmethod of production, and regardless of whether they exist infacilities, in munitions, weapons, or elsewhere.

Small particle size nanoemulsion compositions are useful, for example,as therapeutics for humans or animals, for decontaminating surfaces,individuals or locations colonized or otherwise infected by pathogenicmicroorganisms, for prophylaxis, treatment, and vaccine compositions,for decreasing the infectivity of pathogenic microorganisms infoodstuffs, and the like. The inactivation of a broad range ofpathogenic microrganisms, including, for example, vegetative bacteriaand enveloped viruses and bacterial spores, combined with low toxicity,make small particle size nanoemulsions well-suited for use as a generaldecontamination agent before a specific pathogen is identified.

A. Nanoemulsion Compositions

Particle size reduction to produce a small particle size nanoemulsionfrom a standard emulsion is efficiently and economically accomplished byhigh-pressure homogenizer or microfluidizer. Small particle sizenanoemulsions can be rapidly produced in large quantities and are stablefor many months at a broad range of temperatures.

An emulsion is a composition containing an aqueous phase and an oilphase. The term “emulsion” refers to, without limitation, anyoil-in-water dispersions or droplets, including lipid structures thatcan form as a result of hydrophobic forces that drive apolar residues(e.g., long hydrocarbon chains) away from water and polar head groupstoward water, when a water immiscible phase is mixed with an aqueousphase. These other lipid structures include, but are not limited to,unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles,and lamellar phases. Classical or standard emulsions comprise lipidstructures having an average particle size of greater than about 5 μm indiameter. Standard nanomulsions having smaller particle sizes are known,and comprise lipid structures having an average particle diameter ofabout 500 nm to about 5 μm. In one embodiment, a standard nanoemulsionhas an average particle size of about As used herein, “small particlesize nanoemulsions” refers to emulsions having an average particlediameters of less than or equal to about 250 nm. In one embodiment,average particle diameter is less than or equal to about 200 nm, lessthan or equal to about 150 nm, less than or equal to about 100 nm, orless than or equal to about 50 nm. As used herein, the term“nanoemulsion” can encompass both standard and small particle sizenanoemulsions.

Emulsion particle size can be determined using any means known in theart, such as, for example, using laser light scattering.

A nanoemulsion composition contains about 5 to about 50 percent byvolume (vol %) of aqueous phase. As used herein, percent by volume (vol%) is based on the total volume of an emulsion or small particle sizenanoemulsion. In one embodiment, the aqueous phase is about 10 to about40 vol %. In another embodiment, the aqueous phase is about 15 to about30 vol %. The aqueous phase ranges from a pH of about 4 to about 10. Inone embodiment the pH of the aqueous phase ranges from about 6 to about8. The pH of the aqueous phase can be adjusted by addition of an acid ora base such as, for example, hydrochloric acid or sodium hydroxide. Inone embodiment, the aqueous phase is deionized water (hereinafter“diH2O”) or distilled water.

The oil phase of a nanoemulsion contains an oil and an organic solvent.The oil phase of a nanoemulsion contains about 30 to about 90 vol % oil,based on the total volume of the nanoemulsion. In one embodiment, thenanoemulsion contains about 60 to about 80 vol % oil. In anotherembodiment, the nanoemulsion contains about 60 to about 70 vol % oil.The oil phase also contains from about 3 to about 15 vol % of an organicsolvent based on the total volume of the nanoemulsion. In oneembodiment, the nanoemulsion contains about 5 to about 10 vol % of anorganic solvent.

Suitable oils include, but are not limited to, soybean oil, avocado oil,squalene oil, olive oil, canola oil, corn oil, rapeseed oil, saffloweroil, sunflower oil, fish oils, cinnamon bark, coconut oil, cottonseedoil, flaxseed oil, pine needle oil, silicon oil, mineral oil, essentialoil, flavor oils, water insoluble vitamins, and combinations comprisingone or more of the foregoing oils. In one embodiment, the oil is soybeanoil.

Suitable organic solvents include, but are not limited to, organicphosphate solvents, alcohols, and combinations comprising one or more ofthe foregoing solvents. Suitable organic phosphate solvents include, butare not limited to, dialkyl and trialkyl phosphates having one to tencarbon atoms, more preferably two to eight carbon atoms. The alkylgroups of the di- or trialkyl phosphate can all the same or the alkylgroups can be different. In one embodiment, the trialkyl phosphate istri-n-butyl phosphate. Without being held to theory, it is believed thatorganic solvents used in the small particle size nanoemulsions serve tostabilize the nanoemulsion and remove or disrupt the lipids in themembranes of pathogens.

Suitable alcohols include, for example, C₁-C₁₂ alcohols, diols, andtriols, for example glycerol, methanol, ethanol, propanol, octanol, andcombinations comprising one or more of the foregoing alcohols. In oneembodiment, the alcohol is ethanol or glycerol, or a combinationsthereof.

Small particle size nanoemulsion compositions can also contain one ormore surfactants, present in the aqueous phase, the oil phase, or bothphases of a nanoemulsion. While not limited to any particular proposedmechanism, a nanoemulsion composition may function to remove proteinsfrom bacterial membranes, such that a surfactant that will “strip” amembrane of its proteins may be useful. A nanoemulsion can contain about3 to about 15 vol % of surfactant, based on the total volume ofnanoemulsion. In one embodiment, the nanoemulsion contains about 5 toabout 10 vol % of surfactant.

Suitable surfactants include, but are not limited to, a variety of ionicand nonionic surfactants, as well as other emulsifiers capable ofpromoting the formation of nanoemulsions. Surfactants that allow the oilphase to remain suspended in the water phase can be used. In oneembodiment, the nanoemulsion comprises a non-ionic surfactant such as apolysorbate surfactant, i.e., polyoxyethylene ether. Other usefulsurfactants include, but are not limited to, the polysorbate detergentssold under the tradenames TWEEN® 20, TWEEN® 40, TWEEN® 60, TWEEN® 80,phenoxypolyethoxyethanols and polymers thereof, such as Triton® (i.e.,X-100, X-301, X-165, X-102, X-200), Poloxamer® 407, Spans (20, 40, 60,and 80), tyloxapol, and combinations comprising one or more of theforegoing surfactants. Additional appropriate surfactants includeBrij®30, Brij®35, Brij®52, Brij®56, Brij®58, Brij®72, Brij®76, Brij®78,Brij®92, Brij®97, Brij®98, and Brij® 700. Anionic surfactants include,but are not limited to sodium dodecyl sulfate (SDS). Mixtures ofsurfactants are also contemplated. In one embodiment, the surfactant isTWEEN® 20 or Triton® X-100 or a combination thereof. Triton X-100 is astrong non-ionic detergent and dispersing agent widely used to extractlipids and proteins from biological structures. It also has virucidaleffect against a broad spectrum of enveloped viruses. In anotherembodiment, the surfactant is nonoxynol-9.

Nanoemulsion compositions can further contain various additives.Exemplary additives include, for example, activity modulators, gellingagents, thickeners, auxiliary surfactants, other agents that augmentcleaning and aesthetics, and combinations comprising at least one of theforegoing, so long as they do not significantly adversely affect theactivity and/or stability of the emulsions. Additives can beincorporated into the nanoemulsion or formulated separately from thenanoemulsion, i.e., as a part of a composition containing ananoemulsion.

“Activity modulators” are additives that affect the activity of ananoemulsion against the target microorganism. Exemplary activitymodulators are interaction enhancers such as germination enhancers,therapeutic agents, buffers, and the like, which are described below.

One class of activity modulators thus includes “interaction enhancers,”compounds, or compositions that increase the interaction of thenanoemulsion with the cell wall of a bacterium (e.g., a Gram positive ora Gram negative bacteria) or a fungus, or with a virus envelope. Again,without being bound by theory, it is proposed that the activity of theemulsions is due, in part, to the interaction of a nanoemulsion with amicroorganism membrane or envelope. Suitable interaction enhancersinclude compounds that increase the interaction of the nanoemulsion withthe cell wall of Gram negative bacteria such as Vibrio, Salmonella,Shigella, Pseudomonas, Escherichia, Klebsiella, Proteus, Enterobacter,Serratia, Moraxella, Legionella, Bordetella, Helicobacter, Haemophilus,Neisseria, Brucella, Yersinia, Pasteurella, Bacteiods, and the like.

One exemplary interaction enhancer is a chelating agent. Suitablechelating agents include ethylenediaminetetraacetic acid (EDTA),ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), and combinationsthereof. Chelating agents can be prepared in water or in a buffer, suchas, for example, TRIS buffer. Chelating agents can be premixed with theaqueous phase or can be added to a diluent. Chelating agents can be usedat a concentration of about 1 μM to about 50 mM, based on the totalvolume of the nanoemulsion composition. In one embodiment, theconcentration of the chelating agent is between about 100 μM to about 50mM. In a further embodiment, the concentration of chelating agent can begreater than or equal to about 25 μM, greater than or equal to about 50μM, greater than or equal to about 70 μM greater than or equal to about80 μM, greater than or equal to about 100 μM, greater than or equal toabout 1 mM, or greater than or equal to about 2 mM. In an additionalembodiment, the concentration of chelating agent can be less than orequal to about 40 mM, less than or equal to about 27 mM, less than orequal to about 25 mM, less than or equal to about 10 mM, or less than orequal to about 5 mM.

Another exemplary interaction enhancer is a cationic halogen-containingcompound. A cationic halogen-containing compound can be premixed withthe aqueous phase, or it may can be provided in combination with ananoemulsion in a distinct formulation. A cationic halogen-containingcompound can be used at a concentration of about 0.5 to about 7 vol. %,based on the total volume of the nanoemulsion. In one embodiment, acationic halogen-containing compound can be used at a concentration ofabout 0.5 to about 3 vol. %, based on the total volume of thenanoemulsion.

Suitable cationic halogen-containing compounds include, but are notlimited to, cetylpyridinium halides, cetyltrimethylammonium halides,cetyldimethylethylammonium halides, cetyldimethylbenzylammonium halides,cetyltributylphosphonium halides, dodecyltrimethylammonium halides,tetradecyltrimethylammonium halides, alkylbenzyldimethylammonium saltsand combinations comprising one or more of the foregoing compounds.Suitable halides in the cationic halogen-containing compounds includechloride, fluoride, bromide and iodide. In one embodiment, the halide ischloride or bromide. In another embodiment the cationichalogen-containing compound is cetylpyridinium chloride or benzalkoniumchloride or a combination thereof.

A “germination enhancer” enhances the germination of, for example,spores. Suitable germination enhancing agents include nucleosides,α-amino acids, salts and combinations thereof. Useful nucleosidesinclude inosine. Useful α-amino acids include, for example, glycine andthe L-enantiomers of alanine, valine, leucine, isoleucine, serine,threonine, lysine, phenylalanine, tyrosine, and the alkyl estersthereof. Suitable salts, include, for example, sodium chloride, ammoniumchloride, magnesium chloride, calcium chloride, phosphate bufferedsaline (PBS), and potassium chloride. In one embodiment, the germinationenhancer is a mixture of glucose, fructose, asparagine, sodium chloride,ammonium chloride, calcium chloride, and potassium chloride. In anotherembodiment, the germination enhancer is a combination containingL-alanine, inosine, PBS, and ammonium chloride.

Certain growth media contain germination enhancers and buffers. Thus,when testing nanoemulsions for their ability to inactivate spores,addition of germination enhancers may not be required, if the tests areconducted using media containing such germination enhancers. Similarly,the addition of certain growth media to emulsions can enhance sporicidalactivity.

An effective amount of germination enhancer can be readily determined byone of ordinary skill in the art. Nucleosides and amino acids can beused in amounts of about 0.5 mM to about 100 mM. In one embodiment,nucleosides and amino acids are used at a concentration of about 1 mM toabout 50 mM. In another embodiment, nucleosides and amino acids are usedat a concentration of about 0.5 mM to about 5 mM. Salts can be presentin amounts of about 0.5 mM to about 100 mM and PBS can be used atconcentrations of about 0.05× to about 1×.

A germination enhancer can be incorporated into the aqueous phase priorto formation of the nanoemulsion. In one embodiment a germinationenhancer is active at approximately neutral pH. In another embodiment, agermination enhancer can be active between pH of about 6 to about 8.Adjustment of pH of a nanoemulsion composition containing a germinationenhancer can be achieved by any suitable means, such as, for example,dilution of a nanoemulsions in PBS or by preparations of a neutralnanoemulsion or by the addition of hydrochloric acid or sodiumhydroxide.

“Therapeutic agent” refers to an agent that decreases the infectivity,morbidity, and/or rate of mortality associated with a pathogenicmicroorganism when administered to a subject affected by a pathogenicmicroorganism. Suitable therapeutic agents include, for example,antimicrobial agents, antiviral agents, antifungal agents, and the like,and combinations comprising one or more of the foregoing agents. Thereare many antimicrobial agents currently available for use in treatingbacterial, fungal and viral infections. Generally, these agents includeagents that inhibit cell wall synthesis (e.g., penicillins,cephalosporins, cycloserine, vancomycin, bacitracin), imidazoleantifungal agents (e.g., miconazole, ketoconazole and clotrimazole),agents that act directly to disrupt the cell membrane of themicroorganism (e.g., polymyxin and colistimethate and the antifungalsnystatin and amphotericin B), agents that affect the ribosomal subunitsto inhibit protein synthesis (e.g. chloramphenicol, the tetracyclines,erythromycin and clindamycin), agents that alter protein synthesis andlead to cell death (e.g. aminoglycosides), agents that affect nucleicacid metabolism (e.g. the rifamycins and the quinolones),antimetabolites (e.g., trimethoprim and sulfonamides), and the nucleicacid analogues (e.g. zidovudine, gangcyclovir, vidarabine, andacyclovir) which act to inhibit viral enzymes essential for DNAsynthesis. Other useful therapeutic agents include, but are not limitedto antimicrobials such as phenylphenol, propyl paraben andpoly(hexamethylene biguanide) hydrochloride (PHMB).

Optionally, nanoemulsion compositions can be formed into gels by addinga gelling agent. Suitable gelling agents include, for example, hydrogelssuch as, for example, Natrosol® 250H NF (Hercules, Inc. Wilmington,Del.). A hydrogel can be added at concentration of about 0.5 wt % toabout 5 wt %, based on the total volume of the gel. Other suitablegelling agents include, but are not limited to, about 0.05 wt % to about3 wt % cellulose polymer, such as cellulose gum or cationic guarderivatives, and up to about 10 wt % petrolatum, glycerin, polyethyleneglycol, incroquat behenyl TMS, cetyl palmitate, glycerol stearate, andthe like.

A variety of auxiliary surfactants can optionally be used to enhance theproperties of a nanoemulsion composition. The choice of auxiliarysurfactant depends on the desire of the user with regard to the intendedpurpose of the composition and the commercial availability of thesurfactant. In one embodiment, the auxiliary surfactant is an organic,water-soluble surfactant.

Other optional additives such as perfumes, brighteners, enzymes,colorants, detergent builders, suds suppressors, and the like can alsobe used in the compositions to enhance aesthetics and/or cleaningperformance. Detergent builders sequester calcium and magnesium ionsthat might otherwise bind with and render less effective the auxiliarysurfactants or co-surfactants. Detergent builders are particularlyuseful when auxiliary surfactants are used, and when the compositionsare diluted prior to use with hard tap water, especially water having ahardness of, above about 12 grains/gallon.

A nanoemulsion composition can contain a suds suppressor. A sudssuppressor is a low-foaming co-surfactant that prevents excessivesudsing during employment of the compositions on hard surfaces. Sudssuppressors are also useful in formulations for no-rinse application ofthe composition. Concentrations of about 0.5 vol % to about 5 vol % aregenerally effective. Selection of a suds suppressor depends on itsability to formulate in a nanoemulsion composition and the residue aswell as the cleaning profile of the composition. The suds suppressorshould be chemically compatible with the components in a nanoemulsioncomposition and functional at the pH of a given composition. In oneembodiment the suds suppressor or composition containing a sudssuppressor does not leave a visible residue on surfaces on which acomposition is applied.

Low-foaming co-surfactants can be used as a suds suppressor to mediatethe suds profile in a nanoemulsion composition. Examples of suitablesuds suppressors include block copolymers, alkylated primary andsecondary alcohols, and silicone-based materials. Exemplary blockco-polymers include, e.g., Pluronic® and Tetronic® (BASF Company).Alkylated alcohols include those which are ethoxylated and propoxylated,such as, tergitol (Union Carbide) or poly-tergent® (Olin Corp.).Silicone-based materials include DSE (Dow Corning). The suds suppressorscan be incorporated into the composition by any means known in the art.

B. Method of Making Small Particle Size Nanoemulsions

Small particle size nanoemulsions and compositions containing smallparticle size nanoemulsions can be produced by any suitable means. Asmall particle size nanoemulsion can be formed in the first instance orcan be formed from a nanoemulsion having larger particles. For example,a small particle size nanoemulsion can be produced by reducing theparticle size of a classical or standard nanoemulsion (hereinafter“standard nanoemulsion”), to produce a small particle size nanoemulsionwherein the average nanoemulsion particle size is less than about 250nm. In other words, a nanoemulsion having an average particle diameterof greater than about 250 nm is treated in a manner effective to produceparticles having an average diameter of less than or equal to about 250nm. In one embodiment, small particle size nanoemulsion particles havean average diameter of less than or equal to about 200 nm, less than orequal to about 150 nm, less than or equal to about 100 nm, and less thanor equal to about 50 nm.

Methods for the production of a standard nanoemulsion by mixing an oilphase with an aqueous phase are well-known. A nanoemulsion can be formedby blending an oil phase with an aqueous phase on a volume-to-volumebasis ranging from about 1:9 to about 5:1, about 5:1 to about 3:1, orabout 4:1, oil phase to aqueous phase. The oil and aqueous phases can beblended using an apparatus capable of producing shear forces sufficientto form a nanoemulsion such as, for example, a French press or acommercial low shear or high shear mixer. In one embodiment, thestandard emulsions are prepared under conditions of high shear toproduce a nanoemulsion having a substantially uniform particle sizedistribution. In one embodiment, a standard nanoemulsion for use inpreparing a nanoemulsion composition is comprised of particles having anaverage diameter of about 500 nm to about 5 μm, about 500 nm to about 1μm, 400 nm to about 5 μm, 400 nm to about 1 μm, from about 250 nm toabout 5 μm, and from about 250 nm to about 1 μm. To obtain the desiredpH, the pH of the aqueous phase can be adjusted using hydrochloric acidor sodium hydroxide.

Forming a small particle size nanoemulsion from a standard nanoemulsioncan be accomplished, for example, by passing the standard nanoemulsionthough a microfluidizer (Microfluidics Corp., Newton, Mass.) severaltimes at a pressure sufficient to produce a desired particle size. Amicrofluidizer is a homogenizer that operates by pumping a fluid streaminto an interaction chamber. The interaction chamber containsfixed-geometry microchannels that accelerate the fluid stream, resultingin high turbulence, shear, and cavitation. A H230Z (chamber 400 μmupstream of H210Z chamber (200 μm) can be used. Other chamber size andconfigurations (Y or Z) can be used in forming a nanoemulsion using amicrofluidizer. During homogenization, a nanoemulsion can be circulatedthrough a heat exchanger coil or otherwise cooled to keep thetemperature of the nanoemulsion from increasing significantly. In oneembodiment, a standard nanoemulsion is passed though the microfluidizerfor two to five passes at a pressure of about 2,000 to about 10,000 psi.In another embodiment, the pressure is from 3,000 to about 4,000 poundsper square inch. These conditions can vary depending on factors such asstandard nanoemulsion particle size, nanoemulsion composition, anddesired final particle size

Another means of forming a small particle size nanoemulsion is passageof a standard nanoemulsion through a high pressure homogenizer, like anEmulsiFlex® high pressure homogenizer (Avestin, Inc., Ottawa, Canada).The number of passages through the homogenizer as well as the flow ratewill depend on the particle size of the standard nanoemulsion,nanoemulsion composition, and the desired particle size of the resultingsmall particle size nanoemulsion. Operating pressure is independent fromflow rate and will remain at the set value over the process time. In oneembodiment, the operating pressure is from about 2,500 to about 20,000psi. As with the microfluidizing method discussed above, a nanoemulsioncan be cooled using a heat exchanger or other method and thenanoemulsion can be passed though the homogenizer from about two toabout five times. The particle size depends inversely on both the numberof passages and on the operating pressure. See FIG. 5.

In addition to the above described methods, one can produce a smallparticle size nanoemulsion directly, without premixing. The direct useof, for example, either a microfluidizer or a high pressure homogenizer,as described above, can result in a small particle size nanoemulsionwith the properties discussed above for a small particle sizenanoemulsion produced from a premixed standard nanoemulsion.

Small particle size nanoemulsions can have a consistency ranging from asemi-solid cream to a watery liquid similar to skim milk. Creamyemulsions can be used as-is or mixed with water.

A nanoemulsion can be prepared in a diluted or an undiluted form. In oneembodiment a nanoemulsion shows suitable stability in both diluted andundiluted forms. By suitable stability, it is meant that the emulsionsdo not show any signs of separation (oil phase from aqueous phase) forat least 6 months. In another embodiment a nanoemulsion does not showany sign of separation up to about 2 years. In a further embodiment, ananoemulsion does not show any sign of separation for up to about 3years. Settling of the diluted emulsions is an acceptable characteristicand does not indicate separation of an oil phase from an aqueous phase.Settling is due to separation of emulsions from its diluent, not an oilphase separating from an aqueous phase. Such settling is readilyreversed by simple shaking of the nanoemulsion, while separation of theconcentrated emulsions are not reversed by simple mixing, requiringinstead re-emulsification.

The emulsions can also contain a first nanoemulsion emulsified within asecond nanoemulsion, wherein the first and second emulsions can eachcontain an aqueous phase, an oil phase, and a surfactant. The oil phaseof each of the first and second nanoemulsion can contain an oil and anorganic solvent. The first and second nanoemulsion can be the same ordifferent. A nanoemulsion can also contain a first nanoemulsionre-emulsified to form a second nanoemulsion.

One useful parameter for characterizing a nanoemulsion is “zetapotential.” Zeta potential is the electrical potential of a shear plane(an imaginary surface separating a thin layer of liquid that showselastic behavior) bound to a solid surface that shows normal viscousbehavior. The stability of hydrophobic colloids depends, in part, on thezeta potential. Zeta potential of a nanoemulsion can be about −50 mV toabout +50. In one embodiment, the zeta potential of the emulsions can begreater than or equal to about +10 mV. In another embodiment, the zetapotential is greater than or equal to about +20 mV. In a furtherembodiment, the zeta potential of the emulsions can be less than orequal to about +45 mV, less than or equal to about +40 mV or less thanor equal to about +30 mV.

In one embodiment a nanoemulsion, comprising optional therapeuticagents, can be provided in the form of pharmaceutically acceptablecompositions. The terms “pharmaceutically acceptable” or“pharmacologically acceptable” refer to compositions that do not producesignificant adverse, allergic, or other untoward reactions whenadministered to an animal or a human

Compositions for pharmaceutical use typically comprise apharmaceutically acceptable carrier, for example, solvents, dispersionmedia, coatings, isotonic and absorption delaying agents and the like,and combinations comprising one or more of the foregoing carriers asdescribed, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15thEd. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975), andTHE NATIONAL FORMULARY XIV 14th Ed., Washington: American PharmaceuticalAssociation (1975). Suitable carriers include, but are not limited to,calcium carbonate, carboxymethylcellulose, cellulose, citric acid,dextrate, dextrose, ethyl alcohol, glucose, hydroxymethylcellulose,lactose, magnesium stearate, maltodextrin, mannitol, microcrystallinecellulose, oleate, polyethylene glycols, potassium diphosphate,potassium phosphate, saccharose, sodium diphosphate, sodium phosphate,sorbitol, starch, stearic acid and its salts, sucrose, talc, vegetableoils, water, and combinations comprising one or more of the foregoingcarriers. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the emulsions of the presentinvention, their use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

For topical applications, the pharmaceutically acceptable carriers cantake the form of a liquid, cream, foam, lotion, or gel, and mayadditionally comprise organic solvents, emulsifiers, gelling agents,moisturizers, stabilizers, surfactants, wetting agents, preservatives,time release agents, and minor amounts of humectants, sequesteringagents, dyes, perfumes, and other components commonly used inpharmaceutical compositions for topical administration.

C. Methods of Using Nanoemulsion Compositions to Inactivate a PathogenicMicroorganism

Nanoemulsion compositions are particularly useful in applications whereinactivation of pathogenic microorganisms is desired. The terminactivating means killing, eliminating, neutralizing, or reducing thecapacity of a pathogenic microorganism to infect a host on contact.Nanoemulsion compositions are useful for decreasing the infectivity,morbidity, and/or rate of mortality associated with a variety ofpathogenic microorganisms.

A method of inactivating a pathogenic microorganism comprises contactingthe pathogenic microorganism with an amount a nanoemulsion compositionwhich is effective to inactivate the microorganism. The step ofcontacting can involve contacting any substrate which may be or issuspected to be contaminated with a nanoemulsion composition. Bysubstrate it is meant, without limitation any subject, such as a humanor an animal (contact can be in vivo or ex vivo, any article, anysurface, or any enclosure. A pathogenic microorganism can be, withoutlimitation, a bacteria, a virus, a fungus, a protozoan or a combinationthereof.

The step of contacting can be performed for any amount of timesufficient to inactivate a microorganism. In one embodiment,inactivation occurs within about 5 minutes to about 10 minutes afterinitial contact. However, it is understood that when the emulsions areused in a therapeutic context and applied topically or systemically, theinactivation may occur over a longer period of time, for example, 5, 10,15, 20, 25 30, 60 minutes or longer after administration.

The step of contacting can be performed using any appropriate means ofapplication. For example, compositions can be administered by spraying,fogging, misting, exposure to aerosols, wiping with a wet or saturatedcloth or towlette, drenching, immersing.

Nanoemulsion compositions can be used to inactivate vegetative bacteriaand bacterial spores upon contact. Bacteria inactivated by nanoemulsioncompositions can be Gram negative or Gram positive bacteria. Gramnegative bacteria include, for example and without limitation, Vibrio,Salmonella, Shigella, Pseudomonas, Escherichia, Klebsiella, Proteus,Enterobacter, Serratia, Moraxella, Legionella, Bordetella, Gardnerella,Haemophilus, Neisseria, Brucella, Yersinia, Pasteurella, Bacteroids, andHelicobacter. Gram positive bacteria include, for example, and withoutlimitation, Bacillus, Clostridium, Arthrobacter, Micrococcus,Staphylococcus, Streptococcus, Listeria, Corynebacteria, Planococcus,Mycobacterium, Nocardia, Rhodococcus, and acid fast Bacilli such asMycobacterium. In one embodiment, nanoemulsion compositions can be usedto inactivate Bacillus, including, without limitation B. anthracis, B.cereus, B. circulans, B. subtilis, and B. megaterium. Nanoemulsioncompositions can also be used to inactivate Clostridium, e.g., C.botulinum, C. perfringens, and C. tetani. Other bacteria that can beinactivated by a nanoemulsion include, but are not limited to, H.influenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes andV. cholerae (classical and Eltor), and Yersinia, including, Y. pestis,Y. enterocolitica, and Y. pseudotuberculosis. In another embodiment, thebacteria is B. anthracis. In another embodiment, the bacteria isMycobaterium tuberculosis.

Contacting a bacterial spore with a nanoemulsion inactivates the spore.Without being bound to any theory, it is proposed that the sporicidalability of the nanoemulsions is by initiation of germination withoutcomplete reversion to the vegetative form, leaving the spore susceptibleto disruption by the emulsions. Induction of germination usinggermination enhancers such as inosine and L-alanine can result inacceleration of the sporicidal activity of the nanoemulsion, whileinhibition of initiation of germination with D-alanine can delaysporicidal activity. This unique action of a nanoemulsion, which can bebetter in efficiency than 1% bleach, is interesting because Bacillusspores are generally resistant to most disinfectants including manycommonly used detergents. The sporicidal effect can start almostimmediately. In one embodiment the sporicidal effect occurs within 30minutes of contact with a nanoemulsion.

Contacting a nanoemulsion composition with a virus can inactivate avirus. The effect of nanoemulsion compositions on viral agents can bemonitored using any suitable means, such as, for example, plaquereduction assay (PRA), cellular enzyme-linked immunosorbent assay(ELISA), P-galactosidase assay, and electron microscopy (EM). Viruseswhich can be inactivated by contact with a nanoemulsion compositioninclude, without limitation, and virus of the families Baculoviridae,Herpesviridae, Iridoviridae, Poxviridae, “African Swine Fever Viruses,”Adenoviridae, Caulimoviridae, Myoviridae, Phycodnaviridae, Tectiviridae,Papovaviridae, Circoviridae, Parvoviridae, Hepadnaviridae, Cystoviridae,Bimaviridae, Reoviridae, Coronaviridae, Flaviviridae, Togaviridae,“Arterivirus,” Astroviridae, Caliciviridae, Picomaviridae, Potyviridae,Retroviridae, Orthomyxoviridae, Filoviridae, Paramyxoviridae,Rhabdoviridae, Arenaviridae, and Bunyaviridae. In one embodiment, thevirus is herpes, pox, papilloma, corona, influenza, hepatitis, sendai,sindbis and vaccinia viruses, west nile, hanta, and viruses which causethe common cold.

In yet another embodiment, contacting a nanoemulsion with a fungusinactivates the fungus. In one embodiment, the fungus is a yeast, suchas, for example various species of Candida (e.g., Candida albicans) orfilamentous yeast including but not limited to Aspergillus species ordermatophytes such as Trichophyton rubrum, Trichophyton mentagrophytes,Microsporum canis, Microsporum gypseum, and Epiderophyton floccosum, andtypes thereof, as well as others.

The methods and compositions, or components of the methods andcompositions can be formulated in a single formulation, or can beseparated into binary formulations for later mixing during use, as maybe desired for a particular application. Such components canadvantageously be placed in kits for use against microbial infections,decontaminating instruments and the like. Such kits may contain all ofthe essential materials and reagents required for the delivery of theformulations to the site of their intended action as well as any desiredinstructions.

For in vivo use, the methods and compositions may be formulated into asingle or separate pharmaceutically acceptable syringeable composition.In this case, the container means may itself be an inhalant, syringe,pipette, eye dropper, or other like apparatus, from which theformulation may be applied to an infected area of the body, such as thelungs, injected into an animal, or even applied to and mixed with theother components of the kit.

A kit also can include a means for containing the vials in closeconfinement for commercial sale (e.g., injection or blow-molded plasticcontainers into which the desired vials are retained). Irrespective ofthe number or type of containers, the kits also may comprise, or bepackaged with, an instrument for assisting with theinjection/administration or placement of the ultimate complexcomposition within the body of an animal. Such an instrument may be aninhalant, syringe, pipette, forceps, measured spoon, eyedropper, or anysuch medically approved delivery vehicle.

Actual amounts of nanoemulsions and additives in the compositions can bevaried so as to provide amounts effective to inactivate vegetative aswell as sporular microorganisms and pathogens. Accordingly, the selectedamounts will depend on the nature and site for treatment, the desiredresponse, the desired duration of biocidal action, the condition of thesubject being treated, and other factors. A nanoemulsion composition cancomprise, for example, about 0.001% to about 100% nanoemulsion permilliliter of liquid composition. In one embodiment, a nanoemulsioncomposition can contain about 0.01% to about 90% nanoemulsion permilliliter of liquid. These are merely exemplary ranges. A nanoemulsioncomposition can also comprise greater than about 0.25%, about 1.0%,about 5%, about 10%, about 20%, about 35%, about 50%, about 65%, about80%, about 90%, or about 95% of nanoemulsion per milliliter of liquidcomposition.

The small particle size nanoemulsions as described herein are morestable than standard emulsions under a variety of conditions, showingsubstantially no observable separation or settling for up to one month,preferably up to four months, more preferably up to or more than oneyear, up to about 21° C., preferably up to 40° C. Such stability is atno dilution, up to 2.5% dilution, up to 10% dilution, more preferably upto 50% dilution or higher.

The small particle size nanoemulsions perform equal to or better thanstandard emulsions in inactivating a pathogenic microorganism,exhibiting a less than 10% failure rate, preferably a less than 5%failure rate, more preferably a less than 1% failure rate, and mostpreferably a 0% failure rate against pathogens. The invention is furtherillustrated by the following non-limiting examples.

1. Prevention and Treatment of Infection

Nanoemulsion compositions are useful for the prevention and treatment ofinfection. A method of inactivating a pathogenic microorganism comprisescontacting a subject infected with or suspected to be infected with themicroorganism with a nanoemulsion composition comprising an aqueousphase, an oil phase, and one or more surfactants. The oil phasecomprises an oil and an organic solvent, as discussed above. Thenanoemulsion particles have an average diameter of less than or equal toabout 250 nm. In one embodiment, the particles have an average diameterof less than or equal to about 200 nm, less than or equal to about 150nm, less than or equal to about 100 nm, or less than or equal to about50 nm.

The pathogenic microorganism may have systemically infected the subjector on the surface of the subject. Where the microorganism is not on thesubject, the is delivered to the site of infection by any suitablemethod, for example injection, oral administration, suppositories, andthe like. In one embodiment the subject is an animal. In a furtherembodiment, the animal is a human.

Exemplary infected states that can be treated or prevented withnanoemulsions include, but are not limited to, bacterial, fungal,protozoal, and/or viral vaginal infection, sexually transmitted diseases(STDs), skin infections such as, acne, impetigo, athlete's foot,onychomycosis, candidiasis and other acute fungal infections, herpessimplex and zoster and infections associated with psoriasis or otherskin inflammatory diseases. In one embodiment, an infected state isparticularly susceptible to topical treatment. As used herein, “infectedstates” is inclusive of contamination with pathogenic microorganisms,and treatment and prevention of such infected states includes, but isnot limited to, wound decontamination, decontamination of skin, airways,and/or mucosal surfaces (e.g., with anthrax spores, viruses, bacteria,and/or fungi); and the like. Nanoemulsion compositions can also be usedas a surgical irrigant. The emulsions can be used in the personal healthcare industry in deodorants, soaps, body wash, acne/dermatophytetreatment agents, treatments for halitosis, and skin disinfecting.

Nanoemulsions can be used in a variety of combination therapies,particularly those directed to microorganisms. This approach is oftenadvantageous in avoiding the problems encountered as a result ofmultidrug resistance, for example.

In one embodiment, a nanoemulsion can be used in the prevention ortreatment of genital infections. Such sexually transmitted genitalinfections include, but are not limited to genital herpes, humanpapilloma virus (HPV), human immunodeficiency virus (HIV),trichomoniasis, gonorrhea, syphilis, and chlamydia. A nanoemulsion canbe applied to the genitals either before or after sexual intercourse orboth before and after sexual intercourse. In one embodiment, ananoemulsion is introduced into the vagina of a female, at about thetime of sexual. In another embodiment, a nanoemulsion is introduced intothe vagina of a female prior to intercourse. A nanoemulsion can also beadministered to other mucous membranes. Application of a nanoemulsioncomposition to genitalia can be accomplished using any appropriate meansincluding, for example, ointments, jellies, inserts (suppositories,sponges, and the like), foams, and douches.

A nanoemulsion can also be used in the treatment of nonsexuallytransmitted genital infections, such as fungal, protozoan, bacterialinfections. Fungal infections treatable with a nanoemulsion include, butare not limited, to tinea, candida (e.g., Candida albicans). Nonsexuallytreated bacterial infections treatable with a nanoemulation include, butare not limited, nonspecific vaginitis and bacterial vaginitis causedby, for example, Gardnerella vaginalis, Gardneralla mobiluncus, andMycoplasma hominis.

Nanoemulsion compositions can also be used for the prevention andtreatment of respiratory infection. Nanoemulsion compositions can beused to prevent infection by, without limitation, the common cold,influenza, tuberculosis, legionnaire's disease, and acute respiratorysyndrome (SARS). In one embodiment, a nanoemulsion composition isapplied to the respiratory passages using, for example, a nasal spray,such that the spray coats the respiratory passages before exposure tothese pathogens. In another embodiment, this use can substantiallyinactivate or eliminate a respiratory pathogen preventing the pathogenfrom inducing a pathogenic response. The use of a nanoemulsion in theprevention and treatment of a respiratory infection can also stimulatean immunological response against a specific pathogen which can protectfrom further exposure to the same pathogen.

2. Immunogenic Compositions and Vaccine Applications:

A nanoemulsion can be mixed with a microorganism, a recombinant antigen,or a combination thereof to yield an immunogenic composition. Theconcentration of microorganism can range from approximately 10² toapproximately 10¹⁰. The concentration of antigen can range fromapproximately 1 μg to approximately 1 mg, either alone or mixed withother adjuvants which include but are not limited to CpGoligonucleotides. As used herein “immunogenic composition” refers to anycomposition capable of eliciting an immune response. In one embodiment,the immune response results in production of protective antibodies.

An immunogenic nanoemulsion composition can be administered topically onthe skin or on mucosal membranes of a subject. Administration of animmunogenic composition can be performed using any appropriate means andusing any appropriate formulation known in the art.

In one embodiment, a nanoemulsion composition contains substantiallyinactivated B. anthracis. In one embodiment, a nanoemulsion compositioncontains a peptide comprising at least a portion of anthrax protectiveantigen (PA). PA is known to provide protection against infection inconventional vaccines. A vaccine can also contain an attenuated strainof B. anthracis. In one embodiment, PA is isolated from B. anthracisextract. In another embodiment, PA is recombinant PA. B. anthracis PAconcentration can range from about 1 μg to about 1 mg. In oneembodiment, B. anthracis PA concentration ranged from between about 2.3μg to about 30 μg.

In another embodiment, a nanoemulsion contains substantially inactivatedvaccinia for use as can be used as a small pox vaccination. In a furtherembodiment, a nanoemulsion. In a further embodiment, a nanoemulsion canbe used to create a vaccine against influenza virus. In an additionalembodiment, a vaccine composition can contain an inactivated influenzavirus or a portion thereof.

A nanoemulsion composition can contain substantially inactivatedMycobateria tuberculosis or a portion thereof. In one embodiment, theMycobateria tuberculosis-containing nanoemulsion composition isefficacious as a vaccine against tuberculosis.

A nanoemulsion composition can contain substantially inactivatedhepatitis virus or an portion thereof. In one embodiment, thehepatitis-containing nanoemulsion is efficacious as a vaccine againsthepatitis infection. In another embodiment, the hepatitis virus ishepatitis A, hepatitis B, or hepatitis C, or a mixture thereof. In afurther embodiment, the hepatitis virus is hepatitis B.

A nanoemulsion composition can contain substantially inactivated HIV ora portion thereof. In one embodiment, the HIV-containing nanoemulsion isefficacious as a vaccine against HIV infection.

A nanoemulsion composition can be applied to mucosa to prevent infectionby a microorganism both as a prophylaxis and as a broad spectrumimmunogenic composition. This administration results in a broad spectrumprophylactic immunogenic composition which can both prevent infection bya pathogenic microorganism and also cause an immune response against apathogenic microorganism which comes in contact with thenanoemulsion-coated mucosa of a subject. In one embodiment, thisimmunogenic response can provide protection or future protection to thesubject against a microorganism. In another embodiment, themicroorganism causes influenza, tuberculosis, the common cold, SARS orother respiratory diseases.

In one embodiment, a nanoemulsion composition can be administered priorto contact with microorganisms. Upon contact with a pathogenicmicroorganism, the microorganism is inactivated. Thenanoemulsion-inactivated microorganism can stimulate an immune responsein a subject. In other words, a nanoemulsion composition can inactivatea microorganism and also function an as an adjuvant to aid instimulating an immune response against the microorganism or itsantigens. In one embodiment, the immune response results in antibodiescapable of neutralizing a microorganism and thus providing immunologicalprotection of the subject against the microorganism. Examples ofmicroorganisms which can be used in conjunction with a nanomemulsioninclude, but are not limited to bacteria, bacterial spores , viruses,protozoa, and fungi.

3. Decontamination of Medical Devices

Nanoemulsion compositions are useful for decontaminating surfacescolonized or otherwise infected by pathogenic microorganisms. Theseapplications include, for example, disinfecting or sterilizing medicaldevices, contact lenses and the like, particularly when the devices orlenses are intended to be used in contact with a patient or wearer. Asused herein “medical devices” includes any material or device that isused on, in, or through a patient's body in the course of medicaltreatment, whether prophylactic or therapeutic treatment. Medicaldevices include, but are not limited to, such items as implants, forexample urinary catheters, intravascular catheters, dialysis shunts,wound drain tubes, skin sutures, vascular grafts, implantable meshes,intraocular devices, heart valves, and the like; wound care devices, forexample wound dressings, surgical sutures, biologic graft materials,tape closures and dressings, surgical incise drapes, and the like; drugdelivery devices, for example skin patches, mucosal patches and medicalsponges; and body cavity and personal protection devices, for exampletampons, sponges, surgical and examination gloves, toothbrushes, birthcontrol devices such as IUD's and IUD strings, diaphragms and condoms;and the like.

For applications of this type, the compositions may be convenientlyprovided in the form of a liquid or foam, and may be provided withemulsifiers, surfactants, buffering agents, wetting agents,preservatives, and other components commonly found in compositions ofthis type. The nanoemulsion compositions can be impregnated intoabsorptive materials, such as sutures, bandages, and gauze, or coatedonto the surface of solid phase materials, such as staples, zippers, andcatheters to deliver the compositions to a site for the prevention ortherapy.

4. Sterilization and Disinfectant Applications

The present invention is also useful for disinfection and sterilizationfor medical, hospital, ambulance, institutional, educational,agricultural, food processing, and industrial applications.

In one embodiment, a nanoemulsion composition can be used to preventcontamination, disinfect or sterilize other surfaces, including surfacesused in the food industry, for example equipment and areas where food isprocessed, packaged and stored; vehicles; machinery; household surfaces,and other surfaces. For example, a nanoemulsion composition can be usedto eliminate contamination in meat processing plants, particularly ofmicroorganisms such as Listeria monocytogenes, Salmonellae species andEscherichia species by cleaning slaughterhouses or food packagingfacilities on a continual basis with the composition. In addition,nanoemulsion compositions can be formulated into sprays for hospital,food processing and serving facilities, and household uses such ascleaning and disinfecting patient rooms, household appliances, kitchenand bath surfaces, and the like.

When used in liquid form to decontaminate surfaces, the emulsions can beadmixed with an aqueous carrier liquid. The aqueous carrier liquid ispreferably not toxic and is chemically compatible with the inventiveemulsions. The aqueous carrier liquid can comprise solvents commonlyused in hard surface cleaning compositions. Such solvents are preferablychemically stable at the pH of the emulsions, have good filming/residueproperties, and are miscible with water. Preferred carrier liquidscomprise water or a miscible mixture of a C₂-C₄ alcohol and water. Thealcohol or glycerol can be used to adjust the viscosity of thecompositions Preferably, the aqueous carrier liquid is water or awater-ethanol mixture containing from about 0 to about 50% ethanol orother solvents. Alternatively, when used to clean hard surfaces, theemulsions may be in the form of a gel, foam, or cream, preferably a gel,and may be provided with emulsifiers, surfactants, buffering agents,wetting agents, preservatives, and other components commonly found incompositions of this type.

A nanoemulsion can also be used for mold remediation for building,equipment, and facilities. Examples of molds include, but are notlimited to Cladosporium, Fusarium, Alternaria, Curvularia, Aspergillus,and Penicillium.

A nanoemulsion composition can also be used in the food industry inpreventing and treating food contaminated with pathogens. Thus, suchcompositions may be used to reduce or inhibit microbial growth orotherwise abrogate the deleterious effects of microbial contamination offood. For example, a nanoemulsion composition can be used to killbacteria and fungus on poultry eggs, fruit, vegetables, and meat. Also,the inclusion of a nanoemulsion compositions within the food productitself would be effective in killing bacteria that may have beenaccidentally contaminated meat or poultry. A nanoemulsion compositioncan be included in juice products to prevent growth of certain fungi,which cause contamination and lead to production of mycotoxins. Forthese applications, the nanoemulsion compositions are applied in foodindustry acceptable forms such as washes, dips, additives,preservatives, or seasonings. The use of media and agents for additives,preservatives, and seasonings that are acceptable in food industry iswell known in the art. Except insofar as any conventional additives,preservatives and seasonings are incompatible with the emulsions, theiruse in preventing or treating food born microbes and their toxicproducts is contemplated. Supplementary active ingredients may also beincorporated into the compositions.

5. Biodefense Applications

Nanoemulsion compositions are also useful for biodefense applications,such as, for example, decontamination of a building, surface, garment,and personnel, and disinfection or sterilization of soil and/orwaterways contaminated with a pathogenic microorganism, for example as aresult of a biological warfare attack.

Nanoemulsion compositions can be delivered and applied fordetoxification and decontamination using any appropriate means. Suchdecontamination procedures are well known to those of skill in the artand may involve simple application of the formulation in the form of aliquid spray or may require a more rigorous regimen. For example,nanoemulsion compositions can be applied by, without limitation,spraying, fogging, misting, exposure to aerosols, wiping with a wet orsaturated cloth or towlette for personal skin decontamination;drenching, immersing, spraying with a hand-held spray bottle orbackpack-mounted spray apparatus, showering, spraying with a curtainspray, pouring, dripping, and bathing in the liquid formulation.Additionally, a nanoemulsion can be deployed in a semi-solid carrier,such as in gels, lotions, creams, and pastes. Deployment can beaccomplished by people, deployed from aircraft, helicopters, trucks,tanks, railroad, boats, bicycle, or by automated systems, includingmobile robots.

Deployment can include applying the formulation to a surface inside ofan industrial setting selected from, for example, a food processingplant, a hospital, an agricultural facility, an institutional building,an ambulance, and a cooking area.

A fog (e.g., aerosols with particulate sizes ranging from 1-30 μm) canbe used to achieve effective decontamination in areas wheredecontamination by a foam would be difficult, if not impossible. Oneexample is the interior of air conditioning ducts. A fog can begenerated at registers and other openings in the duct and travel asignificant distance inside of the duct to decontaminate hard to reachplaces. A relatively automated fog-based decontamination system can beset-up at the scene of an attack. Remotely activated foggers can beplaced inside of a facility and turned on at periodic intervals (from aremote location) to completely decontaminate the facility. This methodgreatly decreases the potential for decontamination personnel to beexposed to a biological warfare agent.

A nanoemulsion can be used to decontaminate wounds contaminated with orsuspected to be contaminated with bacteria, bacterial spores, virus,fungus, protazoa or combinations thereof. In one embodiment the bacteriais B. anthracis. In another embodiment, the virus is smallpox. In afurther embodiment, the bacteria is a Yersinia species.

A nanoemulsion can also be used to decontaminate skin contaminated withor suspected to be contaminated with bacteria, bacterial spores, virus,fungus, protazoa or combinations thereof. In one embodiment the bacteriais B. anthracis. In another embodiment, the virus is smallpox. In afurther embodiment the bacteria is Yersinia species.

A nanoemulsion is also useful for prophylaxis treatment of skin againstbacteria, bacterial spores, virus, fungus, protazoa, or combinationsthereof. In one embodiment the bacteria is B. anthracis. In a furtherembodiment, the bacterial spore is cutaneous B. anthracis spore. Inanother embodiment, the virus is smallpox. In a further embodiment, thebacteria is Yersinia species.

A nanoemulsion is also useful for battlefield prophylaxis treatment ofmucosa against bacteria, bacterial spores, virus, fungus, protazoa orcombinations thereof. In one embodiment the bacteria is B. anthracis. Ina further embodiment, the bacterial spore is B. anthracis spore. Inanother embodiment, the virus is smallpox. In a further embodiment, thebacteria is Yersinia species. In one embodiment, nanoemuslions can beapplied intranasally prior to and/or immediately after suspectedcontamination by bacteria, bacterial spores, virus, fungus, orcombinations thereof.

A nanoemulsion composition is also useful for decontamination ofsurfaces contaminated by or suspected to be contaminated by bacteria,bacterial spores, virus, fungus, protazoa or combinations thereof as theresult of, for example, a biological warfare attack. In one embodimentthe bacteria is B. anthracis. In a further embodiment, the bacterialspore is B. anthracis spore. In another embodiment, the virus issmallpox. In a further embodiment, the bacteria is Yersinia species. Inone embodiment, a nanoemulsion composition can be applied prior toand/or immediately after suspected contamination by bacteria, bacterialspores, virus, fungus, or combinations thereof. In a further embodiment,the nanoemulsion composition is applied intranasally. Nanoemulsion canbe applied to surfaces using any appropriate means. In one embodiment, ananoemulsion is delivered as a spray, liquid, fog, foam, or aerosol tocontaminated or suspected contaminated surfaces.

A nanoemulsion composition is also useful for decontamination buildingscontaminated by or suspected to be contaminated by bacteria, bacterialspores, virus, fungus, or combinations thereof. In one embodiment thebacteria is B. anthracis. In a further embodiment, the bacterial sporeis B. anthracis spore. In another embodiment, the virus is smallpox. Ina fuirther embodiment, the virus is Yersinia species. In one embodiment,nanoemuslions can be applied intranasally prior to and/or immediatelyafter suspected contamination by bacteria, bacterial spores, virus,fungus, or combinations thereof. Nanoemulsion can be applied to surfacesusing any appropriate means. In one embodiment, a nanoemulsion isdelivered as a spray, liquid, fog, foam, or aerosol to contaminated orsuspected contaminated surfaces.

EXAMPLE 1 Comparison of Standard Emulsions and Small pParticle SizeNanoemulsions

The nanoemulsions are described by the components of the nanoemulsionaccording to Table 1. Unless otherwise noted, the oil is soybean oil. Inthe formulations, the detergent is listed first, followed by the volumepercentage of the detergent (e.g., W₂₀5 refers to 5 vol % of Tween 20).In the formulations, the designation L2 refers to a small particle sizenanoemulsion produced by a microfluidizer, while the absence of the L2designation refers to a standard nanoemulsion (i.e., average particlesizes of 250 nm to about 1 micrometer). The designation L3 refers tonanoemulsions produced using an Avesting high pressure homogenizer.TABLE 1 Component Symbol Tween 20 W₂₀ Ethanol E Cetylpyridinium chlorideC EDTA ED Triton X-100 X Tributyl phosphate P Glycerol G Benzalkoniumchloride BA

A first nanoemulsion is produced from a mixture containing 548milliliters of water, 2.24 grams of EDTA, 25 grams of cetylpyridiuniumchloride, 125 milliliters of Tween 20, 200 milliliters of ethanol and1600 milliliters of soybean oil. The first nanoemulsion is pre-mixedwith a Silverson L4RT mixer and a fine emulsifier screen for 10 minutesat 10,000±500 revolutions per minute.

The first nanoemulsion is then processed in a Microfluidics M-110EHmicrofluidizer processor using an H210Z (200 μm) chamber downstream ofan H230Z (400 μm) chamber. The first nanoemulsion is passed through themicrofluidizer 3 to 4 times at a pressure of 3,500±500 pounds per squareinch (psi) using cooling ice in the tray surrounding the chambers. Thesmall particle size nanoemulsion produced is referred to as W₂₀EC ED L2.

The second nanoemulsion is then diluted with distilled water to producea series of diluted nanoemulsions. The water and the nanoemulsion can bemixed by shaking, for example, until the nanoemulsion is incorporatedinto the water. Exemplary diluted nanoemulsions are as shown in Table 2.The percentage shown refers to the volume percentage of the nanoemulsionin the dilution. TABLE 2 Formulation water W₂₀5EC ED L2 50% W₂₀5EC ED L2500 mL 500 mL 20% W₂₀5EC ED L2 800 mL 200 mL 10% W₂₀5EC ED L2 900 mL 100mL 5% W₂₀5EC ED L2 950 mL  50 mL 2.5% W₂₀5EC ED L2 975 mL  25 mL

EXAMPLE 2 Method of Making a Small Particle Size Nanoemulsion

A standard nanoemulsion (i.e., particles sizes of 250 nm to 5micrometers) is formed as follows. A mixture of 22 vol % distilledwater, 1 wt/vol % cetylpyridinium chloride, 5 vol % Tween 20, 64 vol %soybean oil, and 8 vol % ethanol based on the total volume of themixture is formed. The nanoemulsion is formed by mixing for 5 minutes at10,000±500 revolutions per minute with a Silverson L4RT mixer with astandard mixing assembly and a fine emulsion screen. The standardnanoemulsion is denoted as W₂₀5EC.

A small particle size nanoemulsion is formed by passing the W₂₀5ECnanoemulsion 4 times through a Microfluidics M-110EH microfluidizerprocessor using an H210Z (200 μm) chamber downstream of an H230Z (400μm) chamber. The small particle size nanoemulsion is denoted as W₂₀5ECL2.

After formation, the W₂₀5EC and W₂₀5EC L2 emulsions are diluted withwater for further testing. Particle sizes are determined by ParticleSizing Systems (PSS) Nicomp Model 380. The samples are diluted 1/2000 indistilled water to measure the particle size. The formulations and dataare shown in Table 3. TABLE 3 Formulation Amount of Amount of AverageParticle No. Formulation nanoemulsion water Size, nm 1 W₂₀5EC — — 421.42 50% W₂₀5EC 90 mL 90 mL 454 3 20% W₂₀5EC 36 mL 144 mL 437.5 4 10%W₂₀5EC 18 mL 162 mL 418.8 5 5% W₂₀5EC 9 mL 171 mL 427.4 6 2.5% W₂₀5EC4.5 mL 175.5 mL 470.3 7 W₂₀5EC L2 — — 152 8 50% W₂₀5EC L2 90 mL 90 mL99.3, 219.5* 9 20% W₂₀5EC L2 36 mL 144 mL 144.2 10 10% W₂₀5EC L2 18 mL162 mL 153 11 5% W₂₀5EC L2 9 mL 171 mL 177.8 12 2.5% W₂₀5EC L2 4.5 mL175.5 mL 157.7*When there is wide range of particle sizes (Nicomp reading), twomethods of calculation are used

As shown in Table 3, dilution of the emulsions does not appreciablyaffect the particle size of either the standard nanoemulsion or thesmall particle size nanoemulsion. The average particle size for theW₂₀5EC emulsions is about 400 to about 500 nm (samples 1-6) and for theW₂₀5EC L2 emulsions is about 140 to about 220 nm (samples 7-12).

EXAMPLE 3 Effect of Microfluidizer Chamber Size on the Size of SmallParticle Size Nanoemulsion Particles

A W₂₀5G BA2 nanoemulsion is passed through different combinations ofmicrofluidizer chambers as shown in Table 4. The W₂₀5G BA2 L2 smallparticle size nanoemulsion is made with 1 pass with a Silverson L4RTmixer and 4 passes through a microfluidizer. Combinations of chamberhaving 75, 200, 400 micrometer microchannels are used to determine therelationship between the size of the microchannels and the size of theparticles produced. TABLE 4 First Second chamber, Particle Samplechamber, μm μm size, nm 1 75 100 174 2 100 75 165 3 75 200 185 4 200 75180 5 75 400 211 6 400 75 199

As shown in Table 4, the chamber size utilized in the microfluidizer,when varied between 75 and 400 μm, does not significantly affect theparticle size of the emulsions. In all cases, the particle size is lessthan or equal to about 250 nm.

EXAMPLE 4 Effect of Number of Passes Through the Microfluidizer onEmulsion Particle Size

A W₂₀5G BA2 nanoemulsion is formed using either a Silverson L4RT mixer(high shear) or a household hand mixer (low shear). The nanoemulsion isthen passed through the microfluidizer for 1 to 6 passes and theparticle size measured. The relationship between the number of passes inthe microfluidizer and the particle size of the emulsions are shown inTable 5 and FIG. 5. TABLE 5 Nanoemulsion Particle Size (nm) (threeindependent Number of Passes experiments Type of First Through withdifferent Sample Mixer Microfluidizer emulsion lots) 1 High shear 1 183,221, 267 2 High shear 2 183, 205, 195 3 High shear 3 210, 202, 201 4High shear 4 155, 156, 156 5 High shear 4 220, 157, 180 6 High shear 5157, 132, 158 7 High shear 6 196, 161, 168 8 Low shear 0 426, 529, 522 9Low shear 1 275, 210, 205 10 Low shear 2 218, 168, 218 11 Low shear 3183, 151, 129 12 Low shear 4 182, 179, 180

As shown in Table 5 and FIG. 5, the number of passes through themicrofluidizer does not have a large effect on the nanoemulsion particlesize. As shown in Sample 4 and 5, 4 passes through the microfluidizerproduces particle sizes consistently below 250 nm. Regarding high shearversus low shear mixing of the starting emulsion, while high shearmixing can produce a more consistent particle size distribution than thelow shear mixing, high shear mixing of the starting emulsion is notrequired to produce the small particle size nanoemulsions.

EXAMPLE 5 Combined Effects of Number of Passes Through theMicrofluidizer and Microfluidizer Chamber Size

The effect of both the number of passes through the microfluidizer andthe chamber size in the microfluidizer are studied for differentformulations. The starting emulsions are prepared using either aSilverson L4RT mixer (“Silv”) or a Ross HSM-410X high shear mixer with a3 inch X-series rotor/stator pre-set to a 0.010 gap (Ross) in order todetermine the effect of mixing method on the particle size of thestarting nanoemulsion (i.e., prior to passage through themicrofluidizer). The L2 emulsions are produced by passing a standardnanoemulsion produced by Silverson mixing through a microfluidizer. Theparticle sizes are shown in Table 6. TABLE 6 Interactive Number Highshear chamber of Particle size, Sample Formulation Mixer type usedpassages nm 1 Nanowash + alcohol* Silv — — 410-486 2 W₂₀5G BA2 Silv, 5 —— 304-371 minutes mixing 3 W₂₀5G BA2 Silv, 20 min — — 283-340 mixing 4S8G Silv — — 350 5 W₂₀5EC Silv — — 381 6 W₂₀5G Silv — — 486 7 W₂₀5G BA2Ross — 1 260 8 W₂₀5G BA2 Ross 2 247 9 W₂₀5G BA2 Ross 3 281 10 W₂₀5G BA2Ross 4 229-254 11 W₂₀5G BA2 Microfluidizer 400, 200 2 196 12 W₂₀5G BA2Microfluidizer 400, 200 3 195 13 W₂₀5G BA2 Microfluidizer 200, 200 3 17314 W₂₀5G BA2 Microfluidizer 75, 200 3 210 15 W₂₀5G BA2 Microfluidizer75, 200 3 235 16 W₂₀5G BA2 Microfluidizer 200, 400 3 179 then dilutedusing 75, 200 17 S8G** Microfluidizer 75, 200 3 161 18 W₂₀5ECMicrofluidizer 75, 200 3 178 19 W₂₀5EC Microfluidizer 75, 200 3 158 20W₂₀5G Microfluidizer 75, 200 3 223 21 W₂₀5GC*** Microfluidizer 400, 2003 189, 200, 225, 226 22 X₈GC Microfluidizer 400, 200 3 130, 145 23X₈E₆G₂**** Microfluidizer 400, 200 3 249*1% W₂₀5 GBA2 + 2 mM EDTA + 20% ethanol**8% SDS, 6% glycerol, 64% soybean oil, 20% water***5% Tween 20, 8% glycerol, 1% cetylpyridinium chloride, 64% soybeanoil, 22% water****8% Triton X100, 6% ethanol, 2% glycerol, 64% soybean oil, 20% water

As shown in Table 6, the Silverson high shear mixer (samples 1-6)produces particle sizes of about 300 nm to about 500 nm. The Ross highshear mixer (Samples 7-10) produces particle sizes of 260 nm after 1pass to about 229 to 254 nm after 4 passes. The Ross high shear mixer isthus capable of producing smaller particle sizes than the Silversonmixer. Also shown in Table 6 is that the samples passed through themicrofluidizer (samples 11-23) have smaller particle sizes than thesamples mixed with either high shear mixer (samples 1 -10).

Regarding the samples passed through the microfluidizer, as shown insamples 11 and 12, similar particle sizes are obtained with either 2 or3 passes through the microfluidizer. Samples 13-16 show that changingthe microchannel size of the microfluidizer chamber does not decreasethe particle size of the emulsions. Samples 17-23 illustrate that,independent of the formulation of the emulsions, emulsions havingparticle sizes of less than about 250 nm can be formed by passing theemulsions through a microfluidizer.

EXAMPLE 6 Particle Sizes and Zeta Potentials for Different NanoemulsionFormulation

In this experiment, the particle sizes and zeta potentials for differentsmall particle size nanoemulsion formulations are determined. Theemulsions are formed by passing a starting nanoemulsion through themicrofluidizer for 3 passes using the H230Z+H210Z chambers. The particlesize and zeta potential are measured by Nicomp 380 Particle sizer. Thedata are shown in Table 7. TABLE 7 Sample Formulation Particle Size Zeta(mV) 1 1% W₂₀5G BA2 L2 + 2 mM EDTA 186 11 2 W₂₀5G BA2 L2 in water 183 273 W₂₀5GC L2 168-236 30-33 4 W₂₀5G SA2 OA2 L2* 226 33 5 W₂₀5E SA3 L2 15431 6 W₂₀5E SA3 L2 + 2 mM EDTA 131 12 7 W₂₀5G SA3 L2** 215 32 8 W₂₀5G SA3L2 + 2 mM EDTA 187, 191 12 9 W₂₀5E L2 189 −25 10 W₂₀5EC L2, premixed156, 182 31 11 W₂₀5EC L2 146 41*5% Tween 20, 8% glycerol, 2% sterylamine, 2% oleyl alcohol, 61% soybeanoil, 21% water**5% Tween 20, 8% glycerol, 3% Sterylamine, 61% Soybean oil, 23% water

As shown in Table 7, all of the formulations have particle sizes of lessthan or equal to about 250 nm.

EXAMPLE 7 Stability of Nanoemulsions

A W₂₀5EC nanoemulsion was formed containing 5% Tween-20, 8% ethanol, 1%cetylpyridinium chloride, 64% soybean oil, and the balance water. AW₂₀5EC L2 nanoemulsion is formed using 2 passes on a microfluidizer. AW₂₀5GC nanoemulsion is formed containing 5% Tween-20, 8% glycerol, 1%cetylpyridinium chloride, 64% soybean oil, and the balance water. AW₂₀5GC L2 nanoemulsion is formed using 2 passes on a microfluidizer. AnX8P nanoemulsion is formed using 8% Triton X-100, 8% tributyl phosphate,and the balance water.

Stability is determined by evaluating the physical appearance of theemulsions. As used herein, creaming is the presence of a white layer ofcreamy material on top of the nanoemulsion that is more opaque than therest of the nanoemulsion. Settling is a gradual decrease in opacity ofthe nanoemulsion from top to bottom due to separation of the more densediluent (water) at the bottom from the less dense nanoemulsion at thetop. The water appears as transparent layer at the bottom of the vial.Settling is classified as follows: Mild settling: the nanoemulsionappears cloudy with a gradient of “cloudiness” where it gets more opaqueas you go upwards. Moderate settling: a partially clear aqueous solutionappears on the bottom of the sample. The rest of the nanoemulsionappears cloudy with a gradient of cloudiness getting more opaque as yougo up. Some creaming may be on the surface. Severe settling:nanoemulsion has the appearance of three distinct layers, a partiallyclear bottom, cloudy middle, and creamy top. Extreme settling: only twolayers, a thick partially clear bottom and a thin creamy top.

Separation is the phase separation of the nanoemulsion ingredients.Separation is classified as follows: Mild separation: the surface of thenanoemulsion shows few visible oil droplets. Moderate separation: thesurface of the nanoemulsion has a film of oil. The bottom of thenanoemulsion may have a clear aqueous layer. Severe separation:nanoemulsion has the appearance of three distinct layers, a clearaqueous layer on the bottom, a white or cloudy middle layer and a denseoily layer on the top. Extreme separation: total separation into an oillayer on top and water on bottom.

The ambient storage stability test includes storing the neat emulsionsin polypropylene bottles or centrifuge tubes at room temperature (22-25°C.). Containers may be mixed or opened during the observation period.The emulsions are observed for separation or any other changes inappearance. The observation period is varied due to differentmanufacturing dates of the emulsions. The data for W₂₀5EC emulsions areshown in Table 8. TABLE 8 Days in Bottle Type of Sample storage fullnesscontainer Appearance 1 579 ¼ 125 ml PP severe separation 93%: <7%nanoemulsion between oil & water 2 619 ¼ 125 ml PP extreme separation 3505 ⅔ 250 ml PP moderate separation- 6% oil 4 585 ⅔ 250 ml PP moderateseparation- 8% oil 5 457 ⅔ 250 ml PP mild separation- 1% oil 6 497 ⅔ 250ml PP moderate separation- 1.5% oil 7 184 full 125 ml PP mild-oil dropin air space 8 224 full 125 ml PP mild-oil drop in air space 9 184 ¾ 125ml PP mild separation- 1% oil film 10 224 ¾ 125 ml PP moderateseparation- 2% oil film 11 184 ⅔ 125 ml PP mild separation- 4% oil 12224 ⅔ 125 ml PP moderate separation- 6% oil 13 112 ¼ 500 ml PP intact 14152 ¼ 500 ml PP moderate separation—3% oil 15 33 full  30 ml PP intact16 74 full  30 ml PP mild separation- 1 of 4 vials with oil film 17 74 ½250 ml PP mild separation*PP = polypropylene

The data for W₂₀5EC L2 emulsions are shown in Table 9. TABLE 9 Days inBottle Type of Sample storage fullness container Appearance 18 116 full 30 ml PP intact 19 157 full  30 ml PP intact 20 74 ¼  60 ml PP intact21 115 ¼  60 ml PP intact 22 75 full 500 ml PP intact 23 115 full 500 mlPP intact 24 33 full  30 ml PP intact 25 74 full  30 ml PP intact

As shown in Tables 8 and 9, the small particle size nanoemulsions aremore stable at room temperature than comparable standard emulsions.Batches of standard W₂₀5EC neat nanoemulsion stored at ambienttemperatures longer than 5 months show oil forming a film or layer onthe surface of the nanoemulsion. The thickness of the oil layer isvariable and may be related in part to the amount of air in the storagecontainer in addition to the number of times the container has beenentered.

Batches of smaller particle size W₂₀5EC L2 neat nanoemulsion are storedat ambient temperatures for up to 4 months. No settling or separation isobserved in these batches.

Accelerated stability testing is also performed as follows. Glass vialsare filled with 20 milliliters of neat, 10% diluted and 2.5% dilutednanoemulsion. The emulsions are stored at 55° C. and observed 3 times aweek for changes in physical apperrance. One additional set of vials forthe W₂₀5EC L2 emulsions is filled completely (about 25 milliliters) toeliminate air during storage. These full vials are inverted at day 7 tofacilitate observation of creaming and separation.

Neat emulsions (100%) of standard W₂₀5EC and small particle sizenanoemulsion W₂₀5EC L2 under accelerated stability testing at 55° C.show a film of oil separating after 4 and 5 days, respectively (FIG. 1and Table 10). TABLE 10 Average Days Average Days to Mild or to Severeor Moderate Separation Extreme Separation Nanoemulsion Neat 10% 2.50%Neat 10% 2.50% X8P 3 N N 10 N N W₂₀5EC 4.3 N N N N N W₂₀5EC L2 5.3 N N NN N W₂₀5EC L2 full* N N N N N N W₂₀5GC 5.7 N N N N N W₂₀5GC L2 8.7 N N NN NN = No separation

For comparison, the X8P neat nanoemulsion shows signs of instabilitywith a distinct clear aqueous layer on the bottom and a 5% oil layer onthe surface. Neat emulsions of both W₂₀5GC and W₂₀5GC L2 show yellowingof the oil film on the surface of the nanoemulsion, whereas for W₂₀5ECand W₂₀5EC L2, the oil film is colorless. The neat small particle sizenanoemulsions are stable for 1-3 days longer than the standardemulsions.

No diluted nanoemulsion (10% or 2.5%) shows separation of oil after 4weeks observation at 55° C. (Table 10).

Table 11 shows the settling observed for the nanoemulsions afteraccelerated aging. TABLE 11 Average Days Average Days to Mild or toSevere or Moderate Settling Extreme Settling Nanoemulsion Neat 10% 2.50%Neat 10% 2.50% X8P N 3 3 N 10 10 W₂₀5EC N 3 3 N 19 10 W₂₀5EC L2 N 10.6 5N N N W₂₀5EC L2 full* N N 5 N N N W₂₀5GC N 5 3 N 26 19 W₂₀5GC L2 N 10 3N N N

On average, the small particle size nanoemulsions exhibit less oilseparation and less separation of the oil and water layers than thestandard emulsions (Table 10). The small particle size nanoemulsionsexhibit comparable settling and creaming to the standard emulsions whenundiluted and improved stability when diluted to 10% or 2.5% (Table 11).

Settling and creaming are more pronounced in the diluted large particlesize emulsions compared to the diluted emulsions stored at 55° C. (FIGS.2-3, Table 12). The 10% W₂₀5EC nanoemulsion is 83% settled after 4weeks, whereas the 10% W₂₀5EC L2 nanoemulsion is only 9% settled. Theonset of settling occurred later in the smaller particle sizenanoemulsion, within 10 days for 10% W₂₀5EC L2 compared to only 3 daysfor 10% W₂₀5EC. Table 12 shows the creaming and settling of theemulsions.

Table 12 shows the separation and settling of emulsions underaccelerated aging conditions TABLE 12 Separation Settling Neat 10% 2.50%Nanoemulsion Oil Water Cream Settling Cream Settling X8P 9 17 13 86 6 94W₂₀5EC 2 0 14 83 5 94 W₂₀5EC L2 3 0 2 9 2 42 W₂₀5EC L2 full* 0 0 2 <14 228 W₂₀5GC 0.3** 0 13 77 5 93 W₂₀5GC L2 0.7** 0 0 11 2 41

The W₂₀ 5EC L2 nanoemulsion that is stored in vials that are completelyfull show no separation and less settling compared to the samenanoemulsion stored in vials containing an air space (Table 12).Interestingly, the bottom breaks off at the seam at day 10 and day 21for 2 of the full vials of diluted nanoemulsion.

The change in pH after accelerated stability testing is measured. The pHof each nanoemulsion is measured at the beginning and at the end of theaccelerated stability incubation at 55° C. Diluted emulsions aremeasured using a 3-in-1 combination electrode and neat emulsions aremeasured with a semi-micro electrode. The initial pH of the neat W₂₀5EC,and W₂₀5EC L2, W₂₀5GC, and W₂₀5GC L2 emulsions is similar for eachnanoemulsion, ranging from 4.2-4.4. The pH increases with increasingdilution of these nanoemulsion to a pH of 5.6 for the 2.5 % dilutions.After 4 weeks at 55° C., the pH of the neat emulsions remains unchanged,whereas the pH of the diluted emulsions decreases to a value similar tothat of the neat nanoemulsion, (4.0-4.4). In contrast, W₂₀5EC L2incubated in vials that are filled completely, slightly increased in pHafter 4 weeks incubation at 55° C. The difference between the neat anddiluted nanoemulsion is also maintained (FIG. 4).

Additional stress testing is preformed by centrifugation, freezing andautoclaving. In the centrifugation test, neat (100%) and a 10% dilutionof W₂₀5EC L2 nanoemulsion are centrifuged at 1,650×g for 30 minutes atroom temperature, then stored at room temperature for observation. Anadditional sample of the 10% dilution of W₂₀5EC L2 is not centrifugedand is stored at room temperature for comparison. After storage at roomtemperature for 6 weeks, no separation of neat or diluted emulsions isobserved. Only slight creaming is seen in the 10% diluted emulsions withno difference between the centrifuged and uncentrifuged sample.

In the freezing test at −18° C. neat nanoemulsion and a 10% dilution ofW₂₀5EC L2 are placed at −18° C. for 24 hours, and then left at roomtemperature for observation. The neat nanoemulsion W₂₀5EC L2 is frozenat −18° C. for 24 hours then thawed and observed. After 24 hours at roomtemperature no separation is observed in the neat or 10% dilutednanoemulsion. Creaming is observed in the 10% diluted nanoemulsion andno settling were noted.

In the autoclaving test neat W₂₀5EC, W₂₀5EC L2, W₂₀5GC, and W₂₀5GC L2emulsions are placed in a Yamato autoclave for 15 minutes at 121° C.,and then stored at room temperature for observation. Both emulsionscontaining ethanol (W₂₀5EC and W₂₀5EC L2) boiled over in the autoclaveand severe separation is observed immediately after autoclaving. Theemulsions containing glycerol are intact after autoclaving and displayedno separation up to 3 days when stored at room temperature.

EXAMPLE 8 Manufacture of Small Particle Size Nanoemulsions Using a HighPressure Homogenizer

This example demonstrates using a high pressure homogenizer (AvestinEmulsiflex C3) to reduce the particle size of a standard nanoemulsion toparticles having a diameter of 50-150 nm. The size of the nanoemulsionparticles depends on the pressure and number of passages.

First, a standard nanoemulsion containing particles having an averagediameter of 250 nm to 5 micrometers, preferably about 300 nanometer to 1micrometer is formed. The standard nanoemulsion contains 22 vol %distilled water, 1 wt/vol % cetylpyridinium chloride, 5 vol % Tween 20,64 vol % soybean oil, 8 vol % ethanol and 2 mM EDTA, based on the totalvolume of the mixture formed. The nanoemulsion is formed by mixing for 5minutes at 10,000±500 revolutions per minute with a Silverson L4RT mixerwith a standard mixing assembly and a fine emulsion screen. The standardnanoemulsion is denoted as W₂₀5EC ED.

Small particle size nanoemulsions containing particles of various sizesare then formed by passing the standard nanoemulsion through an AvestinEmulsiFlex under different pressures ranging from 3,500-17,000 psi. Thenanoemulsion was passed between 4-5 times under the same conditions. Themachine applies high pressure to push the nanoemulsion through a dynamichomogenizing valve. Table 13 describes the different nanoemulsionparticle size resulting from different passages into the emulsifier.TABLE 13 Passages in the high pressure Pressure Particle size Nameemulsifier (psi) (nm) W₂₀5EC ED None — 277 W₂₀5EC ED L3 1 17,000 111W₂₀5EC ED L3 2 17,000 92 W₂₀5EC ED L3 3 17,000 91 W₂₀5EC ED L3 4 17,00065 W₂₀5EC ED L3 1 3,500 164 W₂₀5EC ED L3 2 3,500 123 W₂₀5EC ED L3 33,500 110 W₂₀5EC ED L3 4 3,500 124 W₂₀5EC ED L3 5 3,500 130

Table 13 and FIG. 5 demonstrate that particle size is inverselydependent on the amount of pressure applied during homogenization aswell as the number of passages to which the nanoemulsion is subjected.

EXAMPLE 9 Testing of Disinfectants Containing the Nanoemulsions

Example 9 compares the efficacy of a standard nanoemulsion versus asmall particle size nanoemulsion (denoted L2) as a disinfectant.

The AOAC (Association of Official Analytical Chemist) dilution test is acarrier-based test. Carriers (i.e., stainless steel cylinders) areinoculated with a test microorganism, dried, exposed to a dilution of adisinfectant product, and cultured to assess the survival of thebacteria. A single test involves the evaluation of 60 inoculatedcarriers contaminated with one microorganism against one product sample.In addition to the 60 carriers, 6 carriers are required to estimatecarrier bacterial load and 6 more are included as extras. Thus, a totalof 72 seeded carriers are required to perform a single test.

A contaminated dried cylinder carrier is added to the medication tubes.Immediately after placing carrier in medication tube, tubes are swirled3 times before placing tube into bath. Ten minutes after each carrier isdeposited into the disinfectant, each carrier is removed from themedication tube with a sterile hook, tapped against the interior sidesof the tube to remove the excess disinfectant, and transferred into theprimary subculture tube containing the appropriate neutralizer (Letheenbroth, 10 mL in 20×150 mm tubes). The subculture tubes are swirled for3-4 seconds. Transfer into the primary subculture tubes should be within±5 seconds of the actual time of transfer (10 minutes). The bacterialcarrier load on at least 2 carriers is assayed.

After a minimum of 30 minutes from when the test carrier was deposited,each carrier is transferred using a sterile wire hook to a secondsubculture tube containing 10 mL of the appropriate neutralizer. Thecarriers are transferred in order, but the intervals do not have to betimed. The tubes are swirled for 3-4 seconds and the subculturesincubated at 37° C. for 48 hours. If the broth culture appears turbid,the result is positive. A negative result is one in which the brothappears clear. Each tube is shaken prior to recording results todetermine the presence or absence of turbidity. The primary andsecondary subculture tubes for each carrier represent a “carrier set.” Apositive result in either the primary or secondary subculture tube isconsidered a positive result for a carrier set.

Gram stains are performed on smears taken from the positive culturetubes. For additional confirmatory tests, a loop of broth is streaked onthe selective media appropriate for the test microorganism and incubatedfor 24 hours at 37° C.

Table 14. Gram staining and culture on selective media required toensure the identity of the microorganism. TABLE 14 S. choleraesuis S.aureus P. aeruginosa Gram stain Gram negative rods Gram positive Gramnegative cocci arranged rods in clusters Selective media MacConkey agarMannitol salt Pseudosel agar agar Morphology on Pale large colonies,Circular, small, Circular, small, selective media agar turning lightfluorescent initially opaque, color. yellow turning colonies.fluorescent green over time. Regular media TSA* TSA TSA*Tryptic soy agar

Table 15 show the results for a W₂₀5G BA2+2 mM EDTA at pH 7.2nanoemulsion and a W₂₀5G BA2 L2+2 mM EDTA at pH 7.2 nanoemulsion withStaphylococcus aureus. TABLE 15 Carriers Total Number of PercentageSample Formulation failed tested experiments failed 1 1% W₂₀5G 16 304 65.26% BA2 + 2 mM EDTA 2 1% W₂₀5G BA2 2 240 4 0.83% L2 + 2 mM EDTA 3 1%W₂₀5G BA2 L2 1 300 6 0.33%

As shown in Table 15, a disinfectant made with the small particle sizenanoemulsions has a lower failure ratio than a standard nanoemulsion.The standard nanoemulsion has a failure rate of about 5%. The smallparticle size nanoemulsions have a failure rate of less than 1%.

Table 16 also shows results obtained for various formulations exposed toStaphylococcus aureus. TABLE 16 Number of No. of Sam- Ex- FailedPercentage ple Formulation periments Cylinders failed 1 1% W₂₀5G BA2 + 2mM 6 304  5.3% EDTA pH 7.2 2 1% W₂₀5G BA2 + 2 mM 9 272 11.4% EDTA pH 8.03 1% W₂₀5G BA2 L2 + 2 mM 4 240 0.83% EDTA pH 7.2 4 1% W₂₀5G BA2 pH 7.2 6300 0.33%

Table 16 demonstrates that the small particle size nanoemulsions(Samples 3 and 4) show greater efficacy against Staphylococcus aureusthan the standard emulsions (Samples 1 and 2).

Table 17 shows the results obtained for various formulations exposed toSalmonella choleraesuis. TABLE 17 Number of No. of Sam- Ex- CylindersPercentage ple Formulation periments tested failed 1 1% W₂₀5G BA2 + 2 mM2 120 0% EDTA pH 7.2 2 1% W₂₀5G BA2 + 2 mM 1 30 0% EDTA pH 8.0 3 1%W₂₀5G BA2 L2 + 2 mM 1 60 0% EDTA pH 7.2 4 1% W₂₀5G BA2 (L₂) pH 7.2 60240 0%

Table 17 demonstrates that the small particle size nanoemulsions(Samples 3 and 4) show similar efficacy against Salmonella choleraesuiscompared to the standard emulsions (Samples 1 and 2). Overall in thedisinfectant test, the small particle size nanoemulsions perform as wellas or better than the standard emulsions.

EXAMPLE 10 Bactericidal Properties of the Nanoemulsions AgainstStaphylococcus aureus

The bactericidal activity of the nanoemulsions is tested using a tuberotation test. In this test, first a culture is prepared by picking onecolony from the stock culture plate of Staphylococcus aureus, streakingfresh TSA and incubating overnight at 37° C. The next morning, onecolony is picked from the agar plate and transferred into 25 mL of TSBin a 50 mL screw-cap tube and incubated at 37° C. on a tube rotator for4-5 hours until the culture becomes turbid. Bacteria grown for 4-6 hoursis added to 10 mL TSB until the culture media becomes slightly turbid.

W₂₀5EC and W₂₀5EC L2 are used as previously described. The emulsions arethen diluted to 2%, 1%, 0.2%, 0.1%, and 0.02% by volume with water.

Bactericidal testing is performed as follows. In 1.7 mL microfuge tubes,0.5 mL cell suspension and 0.5 mL of each of the nanoemulsion dilutionsis mixed and the tubes capped. A positive control containing 0.5 mL ofcell suspension and 0.5 mL of sterile distilled water is prepared inparallel. The tubes are incubated on a tube rotator at 37° C. for 10minutes. Each of the preparations is serially diluted (5 log diluation)in a 96-well plate using PBS. 25 μL from each dilution on is incubatedon TSA at 37° C. overnight. The colonies on the control and test platesare counted. The count on the control plate provides the initialbacterial count. The initial bacteria count is provided as:Initial bacterial count=CFU×40×plate dilutionwhere CFU is the colony forming units per mL. The colonies on each ofthe test plates is counted. Plates having between 20-50 CFU are counted.The report log reduction is provided as:Report Log reduction=Log (count on the control treatment)−Log (count onthe treatment).

Small particle size nanoemulsions have several advantages over standardemulsions. First, the small particle size nanoemulsions can be morestable than the standard emulsions when stored at room temperature or at55° C. The small particle size nanoemulsions are capable of resistingseparation or settling when stored at room temperature for four months.The undiluted small particle size nanoemulsions can take about 1 to 3days longer to exhibit moderate separation than the standard emulsions.The 2.5 % to 10 % diluted small particle size nanoemulsions can takeabout 2 to 7 days longer to exhibit moderate to extreme settling thanthe standard emulsions. In addition, the onset of phase separation inthe small particle size nanoemulsions at 55° C. is later than for thestandard emulsions.

Second, the small particle size nanoemulsions perform equal to or betterthan standard emulsions in inactivating bacteria. In a disinfectanttest, the small particle size nanoemulsions exhibit a less than 1%failure rate against Staphylococcus aureus compared to greater than 5%for a standard nanoemulsion. In the same test, both the and standardnanoemulsions have a 0% failure rate against Salmonella choleraesuis. Ina tube rotation test, the small particle size nanoemulsions have aslightly improved killing compared with the standard emulsions againstStaphylococcus aureus killing activity.

While the invention is described with reference to exemplaryembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention. All references andpublications cited herein are incorporated by reference in theirentireties.

1. A composition comprising a nanoemulsion, the nanoemulsion comprising:an aqueous phase; an oil phase comprising an oil and an organic solvent;and one or more surfactants; wherein the nanoemulsion comprisesnanoemulsion particles having an average diameter of from less than 150nm
 2. The composition of claim 1, wherein the nanoemulsion particleshave an average diameter of less than or equal to about 100 nm or lessthan or equal to about 50 nm.
 3. The composition of claim 1, wherein theorganic solvent comprises a C₁-C₁₂ alcohol, diol, or triol, a dialkylphosphate, a trialkyl phosphate or a combination thereof.
 4. Thecomposition of claim 1, wherein the alcohol comprises ethanol, isopropylalcohol, glycerol or a combination thereof.
 5. The composition of claim2, wherein the trialkyl phosphate is tri-n-butyl phosphate.
 6. Thecomposition of claim 1, wherein the oil comprises soybean oil, mineraloil, avocado oil, squalene oil, olive oil, canola oil, corn oil,rapeseed oil, safflower oil, sunflower oil, fish oils, flavor oils,cinnamon bark, coconut oil, cottonseed oil, faxseed oil, pine needleoil, silicon oil, essential oils, water insoluble vitamins, or acombination thereof.
 7. The composition of claim 6, wherein the oil issoybean oil.
 8. The composition of claim 1, wherein the surfactant is anonionic surfactant.
 9. The composition of claim 8, wherein the nonionicsurfactant is TWEEN® 20, Triton® X-100, nonoxynol-9, or a combinationthereof.
 10. The composition of claim 1, wherein the surfactant is acationic surfactant.
 11. The composition of claim 10, wherein thecationic surfactant is cetyl pyridium chloride, benzalkonium chloride ora combination thereof.
 12. The composition of claim 1 comprising: about5 vol. % to about 50 vol. % of aqueous phase; about 30 vol. % to about90 vol. % of oil phase; and about 3 vol. % to about 15 vol. % ofsurfactant.
 13. The composition of claim 1, further comprising anadditive selected from the group consisting of activity modulators,gelling agents, auxiliary surfactants, and a combination comprising oneor more of the foregoing additives.
 14. The composition of claim 13,wherein the activity modulator is an interaction enhancer, a germinationenhancer, a therapeutic agent, or a combination comprising one or moreof the foregoing enhancers.
 15. The composition of claim 14, wherein thegermination enhancer comprises glucose, fructose, asparagine, sodiumchloride, ammonium chloride, calcium chloride, and potassium chloride.16. The composition of claim 14, wherein the germination enhancercomprises L-alanine, inosine, PBS, and ammonium chloride.
 17. Thecomposition of claim 14, wherein the therapeutic agent is anantimicrobial agent, an antifungal agent, an antiviral agent, ananti-mold agent, an anti-mildew agent, or a combination thereof.
 18. Thecomposition of claim 14, wherein the therapeutic agent is a penicillin,a cephalosporin, cycloserine, vancomycin, bacitracin, miconazole,ketoconazole, clotrimazole, polymyxin, colistimethate, nystatin,amphotericin B, chloramphenicol, the tetracyclines, erythromycin,clindamycin, an aminoglycoside, a rifamycin, a quinolone, trimethoprim,a sulfonamide, zidovudine, gangcyclovir, vidarabine, acyclovir,phenylphenol, propyl paraben, poly(hexamethylene biguanide) or acombination comprising one or more of the foregoing therapeutic agents.19. The composition of claim 1, wherein the composition comprises fromabout 0.01% to about 90% nanoemulsion per milliliter of composition. 20.The composition of claim 1, wherein the composition comprises greaterthan about 0.25%, about 1.0%, about 5%, about 10%, about 20%, about 35%,about 50%, about 65%, about 80%, about 90%, or about 95% nanoemulsionper milliliter of composition.
 21. The composition of claim 1, furthercomprising a pharmaceutically acceptable carrier, an auxiliarysurfactant, a suds suppressor, a detergent builder, or a combinationthereof.
 22. A method of reducing the average nanoemulsion particle sizeof a composition comprising a nanoemulsion, comprising treating ananoemulsion comprising an aqueous phase, an oil phase comprising an oiland an organic solvent, and a surfactant, and having nanoemulsionparticles of an average diameter of greater than or equal to about 250nm, so as to reduce the average diameter of the nanoemulsion particlesto less than 150 nm.
 23. A method of making a nanoemulsion, comprisingpassing a first nanoemulsion comprising an aqueous phase, an oil phasecomprising an oil and an organic solvent, and one or more surfactants,wherein the nanoemulsion particles have an average diameter of greaterthan or equal to about 250 nm, through a microfluidizer or high pressurehomogenizer under conditions effective to reduce the average diameter ofthe nanoemulsion particles to less than 150 nm.
 24. The method of claim22, wherein the ratio of oil phase to aqueous phase is from about 1:9 toabout 5:1, from about 5:1 to about 3:1, or from about 4:1.
 25. Themethod of claim 23, wherein the ratio of oil phase to aqueous phase isfrom about 1:9 to about 5:1, from about 5:1 to about 3:1, or from about4:1.
 26. The method of claims 22, wherein the nanoemulsion particleshave an average diameter of less than or equal to about 100 nm or lessthan or equal to about 50 nm.
 27. The method of claims 23, wherein thenanoemulsion particles have an average diameter of less than or equal toabout 100 nm or less than or equal to about 50 nm.
 28. A method ofinactivating a microorganism, comprising contacting the microorganismwith a composition comprising a nanoemulsion for a time effective toinactivate the microorganism, wherein the nanoemulsion comprises: anaqueous phase; an oil phase comprising an oil and an organic solvent;and one or more surfactants; and wherein the nanoemulsion particles havean average diameter of less than 150 nm.
 29. The method of claims 28,wherein the nanoemulsion particles have an average diameter of less thanor equal to about 100 nm or less than or equal to about 50 nm.
 30. Themethod of claim 28, wherein the composition further comprises anactivity modulator, wherein the activity modulator is an interactionenhancer, a germination enhancer, a therapeutic agent, or a combinationcomprising one or more of the foregoing.
 31. The method of claim 28,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 32. The method of claim 28, wherein the microorganism is abacteria, a fungus, a protozoa, a virus, or a combination of one or moreof the foregoing microorganisms.
 33. The method of claim 32, wherein thebacteria is a vegetative bacteria, a bacterial spore, or a combinationthereof.
 34. The method of claim 32, wherein the bacteria comprises aGram negative bacteria, a Gram positive bacteria, an acid fast bacilli,or a combination thereof.
 35. The method of claim 33, wherein thebacterial spore is B. anthracis.
 36. The method of claim 32, wherein thebacteria comprises comprises B. anthracis, B. cereus, B. circulans, B.megatertium, B. subtilis, C. botulinum, C. tetani, C. perfringens, H.influenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes, V.cholerae, S. aureus, Yersinia species, G. vaginalis, G. mobiluncus, M.hominis, Salmonellae species, Shigellae species, Pseudomonas species,Eschericia species, Klebsiella species, Proteus species, Enterobacterspecies, Serratia species, Moraxella species, Legionella species,Bordetella species, Helicobacter species, Arthobacter species,Micrococcus species, Listeria species, Corynebacteria species,Planococcus species, Nocardia species, Rhodococcus species, Mycobacteriaspecies, or a combination thereof.
 37. The method of claim 32, whereinthe virus belongs to a family selected from the group consisting ofOrthomyxoviridae, Retroviridae, African Swine Fever Viruses,Papovaviridae, Hepadnaviridae, Coronaviridae, Flaviviridae, Togaviridae,Picornaviridae, Filoviridae, Paramyxoviridae, or Rhabdoviridae.
 38. Themethod of claim 37, wherein the Orthomyxovirdae virus is influenzavirus, herpes simplex, herpes zoster, sendai virus, sindbis virus, poxvirus, small pox or vaccinia virus.
 39. The method of claim 37, whereinthe Retroviridae is human immunodeficiency virus, west nile virus, hantavirus, or human papilloma virus.
 40. The method of claim 32, wherein thefungus is a yeast or a filamentous fungus.
 41. The method of claim 40,wherein filamentous fungus is selected from the group consisting of anAspergillus species or a dermatophyte.
 42. The method of claim 41,wherein the dermatophyte is selected from the group consisting ofTrichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis,Microsporum gypseum and Epidermophyton floccosum.
 43. The method ofclaim 32, wherein the fungus comprises Cladosporium, Fusarium,Alternaria, Curvularia, Aspergillus Penicillium or a combinationthereof.
 44. A method of inactivating a pathogenic microorganismcomprising contacting a subject infected with the microorganism with acomposition comprising a nanoemulsion comprising: an aqueous phase; anoil phase comprising an oil and an organic solvent; and one or moresurfactants; wherein the nanoemulsion comprises particles having anaverage diameter less than 150 nm.
 45. The method of claim 44, whereinthe particles have an average diameter of less than or equal to about100 nm or less than or equal to about 50 nm.
 46. The method of claim 44,wherein the subject is a human, an animal, or a plant.
 47. The method ofclaim 44, wherein the composition further comprises an activitymodulator, wherein the activity modulator is an interaction enhancer, agermination enhancer, a therapeutic agent, or a combination comprisingone or more of the foregoing.
 48. The method of claim 44, wherein thecomposition further comprises a pharmaceutically acceptable carrier. 49.The method of claim 44, wherein the microorganism is a bacteria, afungus, a filamentous fungus, a protozoa, a virus, or a combination ofone or more of the foregoing microorganisms.
 50. The method of claim 49,wherein the bacteria is a vegetative bacteria, a bacterial spore, or acombination thereof.
 51. The method of claim 49, wherein the fungus is ayeast.
 52. The method of claim 49, wherein the bacteria comprises a Gramnegative bacteria, a Gram positive bacteria, an acid fast bacilli, or acombination thereof.
 53. The method of claim 50, wherein the bacterialspore is B. anthracis.
 54. The method of claim 49, wherein the bacteriais B. anthracis, B. cereus, B. circulans, B. megatertium, B. subtilis,C. botulinum, C. tetani, C. perfringens, H. influenzae, N. gonorrhoeae,S. agalactiae, S. pneumonia, S. pyogenes, V. cholerae, S. aureus,Yersinia species, G. vaginalis, G. mobiluncus, M. hominis, Salmonellaespecies, Shigellae species, Pseudomonas species, Eschericia species,Klebsiella species, Proteus species, Enterobacter species, Serratiaspecies, Moraxella species, Legionella species, Bordetella species,Helicobacter species, Arthobacter species, Micrococcus species, Listeriaspecies, Corynebacteria species, Planococcus species, Nocardia species,Rhodococcus species, Mycobacteria species, or a combination thereof. 55.The method of claim 49, wherein the virus belongs to a family selectedfrom the group consisting of Orthomyxoviridae, Retroviridae, AfricanSwine Fever Viruses, Papovaviridae, Hepadnaviridae, Coronaviridae,Flaviviridae, Togaviridae, Picomaviridae, Filoviridae, Paramyxoviridae,or Rhabdoviridae.
 56. The method of claim 55, herein the Orthomyxovirdaevirus is influenza virus, herpes simplex, herpes zoster, sendai virus,sindbis virus, pox virus, small pox or vaccinia virus.
 57. The method ofclaim 55, herein the Retroviridae is human immunodeficiency virus, westnile virus, hanta virus, or human papilloma virus.
 58. The method ofclaim 49, wherein the filamentous fungus comprises Aspergillus speciesor dermatophytes such as Trichophyton rubrum, Trichophytonmentagrophytes, Microsporum canis, Microsporum gypseum or Epidermophytonfloccosum.
 59. The method of claim 49 wherein the fungus is selectedfrom the group consisting of Cladosporium, Fusarium, Alternaria,Curvularia, Aspergillus and Penicillium.
 60. A method of preventing aninfected state caused by a microorganism, comprising administering to asubject, either before or after exposure to a microorganism, acomposition comprising a nanoemulsion, wherein the nanoemulsioncomprises: an aqueous phase; an oil phase comprising an oil and anorganic solvent; and one or more surfactants; wherein the nanoemulsioncomprises particles having an average diameter less than 150 nm.
 61. Themethod of claim 60, wherein the infected state is a sexually transmittedgenital infection.
 62. The method of claim 61, wherein the sexuallytransmitted genital infection is selected from the group consisting ofgenital herpes, human papilloma virus, human immunodeficiency virus,trichomoniasis, gonorrhea, syphilis, and Chlamydia.
 63. The method ofclaim 60, wherein the microorganism is selected from the groupconsisting of a pox virus, B. anthracis, and Yersinia species.
 64. Themethod of claim 60, wherein the step of administering comprisesapplication of the composition to the mucosa of a subject.
 65. Animmunogenic composition comprising a nanoemulsion, wherein thenanoemulsion comprises: an aqueous phase; an oil phase comprising an oiland an organic solvent; and a surfactant; and wherein the nanoemulsioncomprises nanoemulsion particles having an average diameter of less thanor equal to 250 nm and a microorganism or a portion thereof.
 66. Theimmunogenic composition of claim 65, wherein the particles have anaverage diameter of less than or equal to about 200 nm, less than orequal to about 150 nm, less than or equal to about 100 nm, or less thanor equal to about 50 nm.
 67. The immunogenic composition of claim 65,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 68. The immunogenic composition of claim 65, wherein themicroorganism or portion thereof comprises at least a portion of abacteria, a fungus, a protozoa, a virus, or a combination of one or moreof the foregoing microorganisms.
 69. The immunogenic composition ofclaim 68, wherein the bacteria is a vegetative bacteria, a bacterialspore, or a combination thereof.
 70. The immunogenic composition ofclaim 68, wherein the bacteria is a Gram negative bacteria, a Grampositive bacteria, an acid fast bacilli or a combination thereof. 71.The immunogenic composition of claim 69, wherein the bacterial spore isB. anthracis.
 72. The immunogenic composition of claim 68, wherein thebacteria is a respiratory transmitted bacteria.
 73. The immunogeniccomposition of claim 68, wherein the virus is selected from the groupconsisting of herpes virus, sendai virus, sindbis virus, vaccinia virus,human immunodeficiency virus, west nile virus, hanta virus, humanpapilloma virus, vaccinia virus, SARS, respiratory transmitted virusesand combinations thereof.
 74. The immunogenic composition of claim 65,wherein the microbial antigen is mixed with the nanoemulsion alone orwith an adjuvant.
 75. The immunogenic composition of claim 74, whereinthe adjuvant is a CpG oligonucleotide.
 76. The immunogenic compositionof claim 65, wherein administration of the immunogenic composition to asubject raises antibodies in the subject capable of neutralizing orinactivating the microorganism.
 77. The immunogenic composition of claim65, wherein the microorganism or portion thereof is a PA of B.anthracis.
 78. A method of vaccinating against a microorganismcomprising administering to a subject the a composition comprising ananoemulsion, wherein the nanoemulsion comprises: an aqueous phase; anoil phase comprising an oil and an organic solvent; and one or moresurfactants; wherein the nanoemulsion comprises particles having anaverage diameter of less than 250 nm, wherein the microorganism isinactivated by the composition and an immunological response by thesubject is elicited.
 79. A kit comprising a composition comprising thenanoemulsion of claim 1, wherein the composition is provided in a singleformulation or a binary formulation, wherein the binary formulation ismixed prior to using the composition.
 80. The kit of claim 79, furthercomprising instructions for using the composition.
 81. The kit of claim79, wherein the composition is formulated for topical administration,said kit further including means for applying the composition topically.